Notebook

2026.3.10

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove the lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primers F₁, F₂, and F₃, reverse primers R₂ and R₃, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.15 μL of gRNA, 0.15 μL of Cas12a protein, 0.25 μL of NE Buffer, and 0.25 μL of DEPC water, resulting in a total volume of 0.8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 °C for 30 min.

RPA Section

Prepare eight-tube strips, label the primer groups as F₁R₂, F₁R₃, F₃R₂, and F₂R₂, and establish positive and negative control groups. Each primer group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of the reverse primer, and 4.39 μL of the corresponding forward primer. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the 8-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.8 μL of the pre-incubated RNP, 0.15 μL of Reporter, 3 μL of MgSO₄, and 3.05 μL of ddH₂O, resulting in a total volume of 7 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the 8-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL of nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the F₁R₂ primer pair is the best.

2026.3.18

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.15 μL of gRNA, 0.15 μL of Cas12a protein, 0.25 μL of NE Buffer, and 0.25 μL of DEPC water, resulting in a total volume of 0.8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the temperature gradients as 37, 38, 39, 40, 41, and 42 ℃, and establish positive and negative control groups. Each temperature-gradient group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.8 μL of the pre-incubated RNP, 0.15 μL of Reporter, 3 μL of MgSO₄, and 3.05 μL of ddH₂O, resulting in a total volume of 7 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, and 50 cycles, establish 6 temperature groups at 37, 38, 39, 40, 41, and 42 ℃, set each cycle to 00:01:00, and enable fluorescence acquisition.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal temperature is 41 ℃.

2026.3.22

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each ratio according to the table below, each with a total volume of 0.8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the RNP ratios as 3:1, 2:1, 1:1, 1:2, and 1:3, and establish positive and negative control groups. Each RNP ratio includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.8 μL of the pre-incubated RNP, 0.15 μL of Reporter, 3 μL of MgSO₄, and 3.05 μL of ddH₂O, resulting in a total volume of 7 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that Cas12a:gRNA=3:1 is optimal.

2026.3.26

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.15 μL of gRNA, 0.15 μL of Cas12a protein, 0.25 μL of NE Buffer, and 0.25 μL of DEPC water, resulting in a total volume of 0.8 μL. The specified final RNP concentrations were achieved by adjusting the initial concentrations. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final RNP concentration gradients as 25, 50, 100, 200, and 300 nM, and establish positive and negative control groups. Each final RNP concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.8 μL of the pre-incubated RNP, 0.15 μL of Reporter, 3 μL of MgSO₄, and 3.05 μL of ddH₂O, resulting in a total volume of 7 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal RNP concentration is 25 nM.

2026.3.30

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.15 μL of gRNA, 0.15 μL of Cas12a protein, 0.25 μL of NE Buffer, and 0.25 μL of DEPC water, resulting in a total volume of 0.8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Mg²⁺ concentration gradients as 5 mM, 10 mM, 15 mM, 20 mM, and 25 mM, and establish positive and negative control groups. Each final Mg²⁺ concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. Prepare the CRISPR mixtures corresponding to each final Mg²⁺ concentration gradient according to the table below, each with a total volume of 7 μL per reaction. After preparation, vortex the mixtures thoroughly and centrifuge the tubes, then dispense the mixtures into the eight-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Mg²⁺ concentration is 20 mM.

2026.4.3

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.15 μL of gRNA, 0.15 μL of Cas12a protein, 0.25 μL of NE Buffer, and 0.25 μL of DEPC water, resulting in a total volume of 0.8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Reporter concentration gradients as 500 nM, 750 nM, 1000 nM, 1500 nM, and 2000 nM, and establish positive and negative control groups. Each final Reporter concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. Prepare the CRISPR mixtures corresponding to each final Reporter concentration gradient according to the table below, each with a total volume of 7 μL per reaction. After preparation, vortex the mixtures thoroughly and centrifuge the tubes, then dispense the mixtures into the eight-tube strips.

Add 1 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 1 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Reporter concentration is 2000 nM.

2026.4.7, 4.9

Note: The repeatability experiment was performed six times in total by two wet-lab team members during three different time periods over two days, and only one of these experimental records is presented here.

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.39 μL of gRNA, 1.17 μL of Cas12a protein, 1.95 μL of NE Buffer, and 0.65 μL of DEPC water, resulting in a total volume of 4.16 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the repeatability groups, and establish positive and negative control groups. Each repeatability group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.16 μL of the pre-incubated RNP, 0.3 μL of Reporter, 3.75 μL of MgSO₄, and 0.93 μL of ddH₂O, resulting in a total volume of 5.14 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2.86 μL of the simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 2.86 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that repeatability is good.

2026.4.12

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.39 μL of gRNA, 1.17 μL of Cas12a protein, 1.95 μL of NE Buffer, and 0.65 μL of DEPC water, resulting in a total volume of 4.16 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the six plasmid concentration gradients as 10⁰-10⁵, and establish positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.16 μL of the pre-incubated RNP, 0.3 μL of Reporter, 3.75 μL of MgSO₄, and 0.93 μL of ddH₂O, resulting in a total volume of 5.14 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the respiratory syncytial virus N gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL ddH₂O to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10⁰, with the 10⁰-10⁵ concentration range used in the experiment.

Add 2.86 μL of the simulated plasmid carrying the respiratory syncytial virus N gene at the corresponding concentration gradient to each positive reaction tube and 2.86 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the sensitivity is 1.307 × 10² copies/μL.

2026.4.15

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, the simulated plasmid carrying the human metapneumovirus type B N gene, the simulated plasmid carrying the SARS-CoV-2 N gene, the simulated plasmid carrying the influenza A virus HA gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.39 μL of gRNA, 1.17 μL of Cas12a protein, 1.95 μL of NE Buffer, and 0.65 μL of DEPC water, resulting in a total volume of 4.16 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips and label the plasmid types as the simulated plasmid carrying the respiratory syncytial virus N gene, the simulated plasmid carrying the human metapneumovirus type B N gene, the simulated plasmid carrying the SARS-CoV-2 N gene, and the simulated plasmid carrying the influenza A virus HA gene, while establishing positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.16 μL of the pre-incubated RNP, 0.3 μL of Reporter, 3.75 μL of MgSO₄, and 0.93 μL of ddH₂O, resulting in a total volume of 5.14 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2.86 μL of the corresponding type of simulated viral plasmid to each positive reaction tube and 2.86 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that specificity is good.

2026.4.18

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, saliva, lysis buffer, MgSO₄, forward primer F₁, reverse primer R₂, the simulated plasmid carrying the respiratory syncytial virus N gene, DEPC water, NE Buffer, LbCas12a, gRNA-RSV, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.39 μL of gRNA, 1.17 μL of Cas12a protein, 1.95 μL of NE Buffer, and 0.65 μL of DEPC water, resulting in a total volume of 4.16 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the ddH₂O dilution group and the saliva-lysis-buffer dilution group, and label the positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 23.22 μL of nuclease-free water, 4.39 μL of reverse primer R₂, and 4.39 μL of the corresponding forward primer F₁. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 0.16 μL of the pre-incubated RNP, 0.3 μL of Reporter, 3.75 μL of MgSO₄, and 0.93 μL of ddH₂O, resulting in a total volume of 5.14 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the respiratory syncytial virus N gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL of saliva-lysis-buffer-water mixture to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10³. The control group uses ddH₂O for plasmid dilution.

Add 2.86 μL of the corresponding simulated plasmid carrying the respiratory syncytial virus N gene to each positive reaction tube and 2.86 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 15 μL, 50 cycles, a reaction temperature of 41 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that resistance to matrix interference is good.

2026.4.27

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primers F₁, F₂, F₃, and F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.3 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.1 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the primer groups as F₁R₅, F₂R₅, F₃R₅, and F₅R₅, and establish positive and negative control groups. Each primer group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer, and 2.52 μL of the corresponding forward primer. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the F₅R₅ primer pair is the best.

2026.5.5

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.3 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.1 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the temperature gradients as 37, 38.5, 40, 41, and 42 ℃, and establish positive and negative control groups. Each temperature-gradient group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, and 50 cycles, establish 5 temperature groups at 37, 38.5, 40, 41, and 42 ℃, set each cycle to 00:01:00, and enable fluorescence acquisition.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal temperature is 42 ℃.

2026.5.8

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each ratio according to the table below, each with a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the RNP ratios as 1:0.8, 1:1, 1:1.2, and 1:1.5, and establish positive and negative control groups. Each RNP ratio includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that Cas12a:gRNA=1:0.8 is optimal.

2026.5.12

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each concentration according to the table below, each with a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final RNP concentration gradients as 50, 100, 150, 200, and 300 nM, and establish positive and negative control groups. Each final RNP concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal RNP concentration is 100 nM.

2026.5.15

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.24 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.16 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Mg²⁺ concentration gradients as 16 mM, 18 mM, 20 mM, 22 mM, and 24 mM, and establish positive and negative control groups. Each final Mg²⁺ concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. Prepare the CRISPR mixtures corresponding to each final Mg²⁺ concentration gradient according to the table below, each with a total volume of 10 μL per reaction. After preparation, vortex the mixtures thoroughly and centrifuge the tubes, then dispense the mixtures into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Mg²⁺ concentration is 24 mM.

2026.5.18

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.2 μL of gRNA, 0.2 μL of Cas12a protein, 0.2 μL of NE Buffer, and 2.4 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Reporter concentration gradients as 250 nM, 500 nM, 750 nM, 1000 nM, and 2000 nM, and establish positive and negative control groups. Each final Reporter concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. Prepare the CRISPR mixtures corresponding to each final Reporter concentration gradient according to the table below, each with a total volume of 10 μL per reaction. After preparation, vortex the mixtures thoroughly and centrifuge the tubes, then dispense the mixtures into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Reporter concentration is 2000 nM.

2026.5.21, 5.23

Note: The repeatability experiment was performed six times in total by two wet-lab team members during three different time periods over two days, and only one of these experimental records is presented here.

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.24 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.16 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the repeatability groups, and establish positive and negative control groups. Each repeatability group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that repeatability is good.

2026.5.26

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.24 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.16 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the five plasmid concentration gradients as 10¹-10⁵, and establish positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the human metapneumovirus type B N gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL ddH₂O to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10¹, with the 10¹-10⁵ concentration range used in the experiment.

Add 2 μL of the simulated plasmid carrying the human metapneumovirus type B N gene at the corresponding concentration gradient to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the sensitivity is 1.43 × 10¹ copies/μL.

2026.5.29

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, the simulated plasmid carrying the respiratory syncytial virus N gene, the simulated plasmid carrying the SARS-CoV-2 N gene, the simulated plasmid carrying the influenza A virus HA gene, the simulated plasmid carrying the human parainfluenza virus HN gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.24 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.16 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips and label the plasmid types as the simulated plasmid carrying the human metapneumovirus type B N gene, the simulated plasmid carrying the respiratory syncytial virus N gene, the simulated plasmid carrying the SARS-CoV-2 N gene, the simulated plasmid carrying the influenza A virus HA gene, and the simulated plasmid carrying the human parainfluenza virus HN gene, while establishing positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the corresponding type of simulated viral plasmid to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that specificity is good.

2026.6.2

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, saliva, lysis buffer, MgSO₄, forward primer F₅, reverse primer R₅, the simulated plasmid carrying the human metapneumovirus type B N gene, DEPC water, NE Buffer, LbCas12a, gRNA-hMPV-B, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.24 μL of gRNA, 0.3 μL of Cas12a protein, 0.3 μL of NE Buffer, and 2.16 μL of DEPC water, resulting in a total volume of 3 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the ddH₂O dilution group and the saliva-lysis-buffer dilution group, and label the positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18.96 μL of nuclease-free water, 2.52 μL of reverse primer R₅, and 2.52 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 8 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 3 μL of the pre-incubated RNP, 2 μL of Reporter, and 5 μL of MgSO₄, resulting in a total volume of 10 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the human metapneumovirus type B N gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL of saliva-lysis-buffer-water mixture to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10³. The control group uses ddH₂O for plasmid dilution.

Add 2 μL of the corresponding simulated plasmid carrying the human metapneumovirus type B N gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 50 cycles, a reaction temperature of 42 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that resistance to matrix interference is good.

2026.6.9

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primers F₁, F₂, and F₈, reverse primers R₁, R₃, and R₅, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the primer groups as F₁R₁, F₈R₁, F₂R₃, and F₂R₅, and establish positive and negative control groups. Each primer group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer, and 3 μL of the corresponding forward primer. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the F₂R₃ primer pair is the best.

2026.6.16

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the temperature gradients as 31, 34, 37, 40, 43, and 46 ℃, and establish positive and negative control groups. Each temperature-gradient group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, and 40 cycles, establish 6 temperature groups at 31, 34, 37, 40, 43, and 46 ℃, set each cycle to 00:01:00, and enable fluorescence acquisition.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal temperature is 40 ℃.

2026.6.19

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each ratio according to the table below, each with a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the RNP ratios as 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3, and establish positive and negative control groups. Each RNP ratio includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that Cas12a:gRNA=3:1 is optimal.

2026.6.23

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. The specified final RNP concentrations were achieved by adjusting the initial concentrations. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final RNP concentration gradients as 20, 60, 100, 140, 180, and 220 nM, and establish positive and negative control groups. Each final RNP concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal RNP concentration is 220 nM.

2026.6.26

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Mg²⁺ concentration gradients as 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM, and establish positive and negative control groups. Each final Mg²⁺ concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR system on a per-reaction basis. Adjust the initial concentration to achieve the specified final Mg²⁺ concentration. Add 8 μL of the incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄ at the corresponding final concentration to each tube to obtain a total volume of 11 μL. After preparation, vortex to mix thoroughly, centrifuge, and dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Mg²⁺ concentration is 75 mM.

2026.6.29, 7.1

Note: The repeatability experiment was performed six times in total by two wet-lab team members during three different time periods over two days, and only one of these experimental records is presented here.

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the repeatability groups, and establish positive and negative control groups. Each repeatability group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that repeatability is good.

2026.7.3

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the five plasmid concentration gradients as 10¹-10⁵, and establish positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL ddH₂O to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10¹, with the 10¹-10⁵ concentration range used in the experiment.

Add 2 μL of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene at the corresponding concentration gradient to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the sensitivity is 1.0 × 10² copies/μL.

2026.7.6

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pyogenes speB gene, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips and label the plasmid types as the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pyogenes speB gene, and the simulated plasmid carrying the Bordetella pertussis IS1663 gene, while establishing positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the corresponding type of simulated bacterial plasmid to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that specificity is good.

2026.7.9

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, saliva, lysis buffer, MgSO₄, forward primer F₂, reverse primer R₃, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, DEPC water, NE Buffer, LbCas12a, gRNA-SPN, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 1.375 μL of gRNA, 4.125 μL of Cas12a protein, and 2.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the ddH₂O dilution group and the saliva-lysis-buffer dilution group, and label the positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₃, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Streptococcus pneumoniae lytA gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL of saliva-lysis-buffer-water mixture to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10³. The control group uses ddH₂O for plasmid dilution.

Add 2 μL of the corresponding simulated plasmid carrying the Streptococcus pneumoniae lytA gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that resistance to matrix interference is good.

2026.7.14

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primers F₁, F₂, F₃, and F₅, reverse primers R₁ and R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the primer groups as F₁R₁, F₂R₁, F₃R₁, and F₅R₂, and establish positive and negative control groups. Each primer group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer, and 3 μL of the corresponding forward primer. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the F₅R₂ primer pair is the best.

2026.7.18

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the temperature gradients as 31, 34, 37, 40, 43, and 46 ℃, and establish positive and negative control groups. Each temperature-gradient group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, and 40 cycles, establish 6 temperature groups at 31, 34, 37, 40, 43, and 46 ℃, set each cycle to 00:01:00, and enable fluorescence acquisition.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal temperature is 40 ℃.

2026.7.20

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each ratio according to the table below, each with a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the RNP ratios as 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3, and establish positive and negative control groups. Each RNP ratio includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that Cas12a:gRNA=2:1 is optimal.

2026.7.22

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. The specified final RNP concentrations were achieved by adjusting the initial concentrations. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final RNP concentration gradients as 20, 60, 100, 140, 180, and 220 nM, and establish positive and negative control groups. Each final RNP concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal RNP concentration is 100 nM.

2026.7.24

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Mg²⁺ concentration gradients as 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM, and establish positive and negative control groups. Each final Mg²⁺ concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR system on a per-reaction basis. Adjust the initial concentration to achieve the specified final Mg²⁺ concentration. Add 8 μL of the incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄ to each tube to obtain a total volume of 11 μL. After preparation, vortex to mix thoroughly, centrifuge, and dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Mg²⁺ concentration is 50 mM.

2026.7.26, 7.28

Note: The repeatability experiment was performed six times in total by two wet-lab team members during three different time periods over two days, and only one of these experimental records is presented here.

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the repeatability groups, and establish positive and negative control groups. Each repeatability group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that repeatability is good.

2026.7.30

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the six plasmid concentration gradients as 10⁰-10⁵, and establish positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Bordetella pertussis IS1663 gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL ddH₂O to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10⁰, with the 10⁰-10⁵ concentration range used in the experiment.

Add 2 μL of the simulated plasmid carrying the Bordetella pertussis IS1663 gene at the corresponding concentration gradient to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the sensitivity is 1.0 × 10¹ copies/μL.

2026.8.1

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, the simulated plasmid carrying the Streptococcus pyogenes speB gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips and label the plasmid types as the simulated plasmid carrying the Bordetella pertussis IS1663 gene, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, and the simulated plasmid carrying the Streptococcus pyogenes speB gene, while establishing positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the corresponding type of simulated bacterial plasmid to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that specificity is good.

2026.8.3

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, saliva, lysis buffer, MgSO₄, forward primer F₅, reverse primer R₂, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-BP, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.84 μL of gRNA, 1.66 μL of Cas12a protein, and 5.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the ddH₂O dilution group and the saliva-lysis-buffer dilution group, and label the positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₅. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Bordetella pertussis IS1663 gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL of saliva-lysis-buffer-water mixture to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10³. The control group uses ddH₂O for plasmid dilution.

Add 2 μL of the corresponding simulated plasmid carrying the Bordetella pertussis IS1663 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that resistance to matrix interference is good.

2026.8.7

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primers R₁, R₂, R₃, and R₄, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the primer groups as F₂R₁, F₂R₂, F₂R₃, and F₂R₄, and establish positive and negative control groups. Each primer group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer, and 3 μL of the corresponding forward primer. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the F₂R₂ primer pair is the best.

2026.8.11

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the temperature gradients as 31, 34, 37, 40, 43, and 46 ℃, and establish positive and negative control groups. Each temperature-gradient group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, and 40 cycles, establish 6 temperature groups at 31, 34, 37, 40, 43, and 46 ℃, set each cycle to 00:01:00, and enable fluorescence acquisition.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal temperature is 40 ℃.

2026.8.13

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. Prepare the RNP systems corresponding to each ratio according to the table below, each with a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixtures at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the RNP ratios as 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3, and establish positive and negative control groups. Each RNP ratio includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that Cas12a:gRNA=3:1 is optimal.

2026.8.15

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. The specified final RNP concentrations were achieved by adjusting the initial concentrations. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final RNP concentration gradients as 20, 60, 100, 140, 180, and 220 nM, and establish positive and negative control groups. Each final RNP concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal RNP concentration is 140 nM.

2026.8.17

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the final Mg²⁺ concentration gradients as 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM, and establish positive and negative control groups. Each final Mg²⁺ concentration gradient includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR system on a per-reaction basis. Adjust the initial concentration to achieve the specified final Mg²⁺ concentration. Add 8 μL of the incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄ to each tube to obtain a total volume of 11 μL. After preparation, vortex to mix thoroughly, centrifuge, and dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the optimal Mg²⁺ concentration is 75 mM.

2026.8.19, 8.21

Note: The repeatability experiment was performed six times in total by two wet-lab team members during three different time periods over two days, and only one of these experimental records is presented here.

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the repeatability groups, and establish positive and negative control groups. Each repeatability group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that repeatability is good.

2026.8.22

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the five plasmid concentration gradients as 10¹-10⁵, and establish positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL ddH₂O to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10¹, with the 10¹-10⁵ concentration range used in the experiment.

Add 2 μL of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene at the corresponding concentration gradient to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that the sensitivity is 1.0 × 10³ copies/μL.

2026.8.24

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, the simulated plasmid carrying the Streptococcus pyogenes speB gene, the simulated plasmid carrying the Bordetella pertussis IS1663 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips and label the plasmid types as the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, the simulated plasmid carrying the Streptococcus pneumoniae lytA gene, the simulated plasmid carrying the Streptococcus pyogenes speB gene, and the simulated plasmid carrying the Bordetella pertussis IS1663 gene, while establishing positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Add 2 μL of the corresponding type of simulated bacterial plasmid to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that specificity is good.

2026.8.26

Turn on the UV lamp in the clean bench and irradiate the work area for 30 min, then turn off the UV lamp. Spray the work surface with nucleic acid remover and wipe it clean. Prepare the experimental reagents. Remove lyophilized pellet tubes, nuclease-free water, saliva, lysis buffer, MgSO₄, forward primer F₂, reverse primer R₂, the simulated plasmid carrying the Haemophilus influenzae omp P6 gene, DEPC water, NE Buffer, LbCas12a, gRNA-HI, and Reporter from the refrigerator and allow them to thaw. Thoroughly vortex all reagents before use.

RNP Section

Prepare the RNP mixture on a per-tube basis. To each tube, add 0.875 μL of gRNA, 2.625 μL of Cas12a protein, and 4.5 μL of NE Buffer, resulting in a total volume of 8 μL. After preparation, centrifuge the tubes and pre-incubate the RNP mixture at 37 ℃ for 30 min.

RPA Section

Prepare eight-tube strips, label the ddH₂O dilution group and the saliva-lysis-buffer dilution group, and label the positive and negative control groups. Each group includes both positive and negative groups, with replicate reactions set up for each.

Prepare the RPA mixture in a single pellet tube. To the pellet tube, add 18 μL of nuclease-free water, 3 μL of reverse primer R₂, and 3 μL of the corresponding forward primer F₂. Vortex thoroughly to mix and centrifuge the tube, then dispense 7 μL of the mixture into each tube of the eight-tube strip.

CRISPR Section

Prepare the CRISPR mixture on a per-tube basis. To each tube, add 8 μL of the pre-incubated RNP, 2 μL of Reporter, and 1 μL of MgSO₄, resulting in a total volume of 11 μL. After preparation, vortex the mixture thoroughly and centrifuge the tube, then dispense the mixture into the eight-tube strips.

Perform a concentration-gradient dilution of the simulated plasmid carrying the Haemophilus influenzae omp P6 gene. The initial plasmid concentration was on the order of 10⁸. Transfer 2 μL into an EP tube, and then add 18 μL of saliva-lysis-buffer-water mixture to prepare the 10⁷ concentration level. Continue serial dilution in the same manner to 10³. The control group uses ddH₂O for plasmid dilution.

Add 2 μL of the corresponding simulated plasmid carrying the Haemophilus influenzae omp P6 gene to each positive reaction tube and 2 μL nuclease-free water to each negative reaction tube.

Set the qPCR program with a heated-lid temperature of 105 ℃, a reaction volume of 20 μL, 40 cycles, a reaction temperature of 40 ℃, and a duration of 00:01:00 per cycle, with fluorescence acquisition enabled.

Place the centrifuged reaction tubes, in which the reaction mixtures have been thoroughly mixed, into the qPCR instrument in sequence.

Turn off the ventilation, spray the workbench with nucleic acid remover, and turn on the UV lamp to irradiate the work area for 30 min. Then, turn off the UV lamp, wipe the workbench, and return all instruments and reagents to their designated positions.

After the experiment, collect the data and perform graphical analysis. The results show that resistance to matrix interference is good.