Protocol

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator; prepare reverse primers R₂ and R₃ and forward primers F₁, F₂, and F₃; thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Divide the reactions into four groups, F₁R₂, F₁R₃, F₃R₂, and F₂R₂. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.1 Optimal Reaction Temperature

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument to six temperature gradients of 37, 38, 39, 40, 41, and 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.2 Optimal RNP Ratio

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare five RNP-ratio groups of 3:1, 2:1, 1:1, 1:2, and 1:3. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Cas12a:gRNA	3:1	2:1	1:1	1:2	1:3
   Cas12a (μL)	0.225	0.200	0.150	0.100	0.075
   gRNA (μL)	0.075	0.100	0.150	0.200	0.225
   DEPC Water (μL)	0.425	0.4	0.25	0.3	0.275
   NE Buffer (μL)	0.075	0.100	0.250	0.200	0.225

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.3 Optimal RNP Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare five RNP concentration groups with final concentrations of 25, 50, 100, 200, and 300 nM. Prepare the RNP systems according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.4 Optimal Mg²⁺ Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare five MgSO₄ groups with final concentrations of 5, 10, 15, 20, and 25 mM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table below. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

   Final Mg²⁺ Concentration	5 mM	10 mM	15 mM	20 mM	25 mM
   RNP (μL)	0.80	0.80	0.80	0.80	0.80
   Reporter (μL)	0.15	0.15	0.15	0.15	0.15
   MgSO₄ (μL)	0.75	1.50	2.25	3.00	3.75
   Nuclease-Free Water (μL)	5.30	4.55	3.80	3.05	2.30

6. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

2.5 Optimal Reporter Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare five reporter groups with final concentrations of 500, 750, 1000, 1500, and 2000 nM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table below. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

   Final Reporter Concentration	500 nM	750 nM	1000 nM	1500 nM	2000 nM
   RNP (μL)	0.80	0.80	0.80	0.80	0.80
   MgSO₄ (μL)	3.00	3.00	3.00	3.00	3.00
   Reporter (100 μM, μL)	0.075	0.1125	0.15	0.225	0.30
   Nuclease-Free Water (μL)	3.125	3.0875	3.05	2.975	2.90

6. Add 1 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 1 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.1 Repeatability

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2.86 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 2.86 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

Note: This experiment should be repeated multiple times by two or more operators during different time periods.

3.2 Sensitivity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare six concentration gradients, from 10⁰ to 10⁵ copies/μL, of the simulated plasmid carrying the respiratory syncytial virus N gene.

6. Add 2.86 μL of the simulated plasmid carrying the RSV N gene at the corresponding concentration gradient to the cap of each positive-reaction tube and 2.86 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.3 Specificity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare the target simulated plasmid carrying the RSV N gene and the non-target simulated plasmids carrying the hMPV-B N gene, SARS-CoV-2 N gene, and IAV HA gene.

6. Add 2.86 μL of the corresponding type of simulated plasmid to the caps of the positive-reaction tubes and 2.86 μL of negative-control water to the caps of the negative-reaction tubes. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.4 Mechanism Interference Resistance

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare two groups of simulated plasmids carrying the RSV N gene. Dilute one group with a saliva-lysis-buffer-water solution and the other group with ddH₂O; dilute both groups to 10³ copies/μL.

6. Add 2.86 μL of the simulated plasmid carrying the RSV N gene to the cap of each positive-reaction tube and 2.86 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 41 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator; prepare reverse primer R₅ and forward primers F₁, F₂, F₃, and F₅; thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Divide the reactions into four groups, F₁R₅, F₂R₅, F₃R₅, and F₅R₅. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.1 Optimal Reaction Temperature

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Module	Component	Volume Added (μL)
   RNP System	gRNA (crRNA)	0.3
   	Cas12a Protein	0.3
   	NE Buffer	0.3
   	DEPC Water	2.1

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument to five temperature gradients of 37, 38.5, 40, 41, and 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.2 Optimal RNP Ratio

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare four RNP-ratio groups of 1:0.8, 1:1, 1:1.2, and 1:1.5. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Cas12a:gRNA	1:0.8	1:1	1:1.2	1:1.5
   Cas12a (μL)	0.3	0.3	0.3	0.3
   gRNA (μL)	0.24	0.3	0.36	0.45
   DEPC water (μL)	2.16	2.1	2.04	1.95
   NE Buffer (μL)	0.3	0.3	0.3	0.3

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.3 Optimal RNP Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare five RNP groups with final concentrations of 50, 100, 150, 200, and 300 nM. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Final RNP concentration	50 nM	100 nM	150 nM	200 nM	300 nM
   Cas12a (μL)	0.1	0.2	0.3	0.4	0.6
   gRNA (μL)	0.1	0.2	0.3	0.4	0.6
   DEPC water (μL)	2.7	2.4	2.1	1.8	1.2
   NE Buffer (μL)	0.1	0.2	0.3	0.4	0.6

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.4 Optimal Mg²⁺ Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Module	Component	Volume Added (μL)
   RNP System	gRNA (crRNA)	0.24
   	Cas12a Protein	0.3
   	NE Buffer	0.3
   	DEPC Water	2.16

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare five MgSO₄ groups with final concentrations of 16, 18, 20, 22, and 24 mM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table below. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

   Final Mg²⁺ Concentration	16 mM	18 mM	20 mM	22 mM	24 mM
   RNP (μL)	3.00	3.00	3.00	3.00	3.00
   Reporter (μL)	2.00	2.00	2.00	2.00	2.00
   MgSO₄ (μL)	3.20	3.60	4.00	4.40	4.80
   Nuclease-Free Water (μL)	1.80	1.40	1.00	0.60	0.20

6. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

2.5 Optimal Reporter Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Module	Component	Volume Added (μL)
   RNP System	gRNA (crRNA)	0.2
   	Cas12a Protein	0.2
   	NE Buffer	0.2
   	DEPC Water	2.4

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare five reporter groups with final concentrations of 250, 500, 750, 1000, and 2000 nM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table below. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

   Final Reporter Concentration	250 nM	500 nM	750 nM	1000 nM	2000 nM
   RNP (μL)	3.00	3.00	3.00	3.00	3.00
   MgSO₄ (μL)	5.00	5.00	5.00	5.00	5.00
   Reporter (100 μM, μL)	0.05	0.10	0.15	0.20	0.40
   Nuclease-Free Water (μL)	1.95	1.90	1.85	1.80	1.60

6. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.1 Repeatability

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

Note: This experiment should be repeated multiple times by two or more operators during different time periods.

3.2 Sensitivity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare five concentration gradients, from 10¹ to 10⁵ copies/μL, of the simulated plasmid carrying the hMPV-B N gene.

6. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene at the corresponding concentration gradient to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.3 Specificity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare the target simulated plasmid carrying the hMPV-B N gene and the non-target simulated plasmids carrying the RSV N gene, SARS-CoV-2 N gene, IAV HA gene, and hPIV HN gene.

6. Add 2 μL of the corresponding type of simulated plasmid to the caps of the positive-reaction tubes and 2 μL of negative-control water to the caps of the negative-reaction tubes. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.4 Mechanism Interference Resistance

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare two groups of simulated plasmids carrying the hMPV-B N gene. Dilute one group with a saliva-lysis-buffer-water solution and the other group with ddH₂O; dilute both groups to 10³ copies/μL.

6. Add 2 μL of the simulated plasmid carrying the hMPV-B N gene to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 42 °C for 50 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator; prepare forward primers F₁, F₂, and F₈ and reverse primers R₁, R₃, and R₅; thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Divide the reactions into four groups, F₁R₁, F₈R₁, F₂R₃, and F₂R₅. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.1 Optimal Reaction Temperature

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument to six temperature gradients of 31, 34, 37, 40, 43, and 46 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.2 Optimal RNP Ratio

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP-ratio groups of 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Cas12a:gRNA	3:1	2:1	1.5:1	1:1.5	1:2	1:3
   Cas12a(μL)	4.125	3.667	3.3	2.2	1.833	1.375
   gRNA(μL)	1.375	1.833	2.2	3.3	3.667	4.125
   NE Buffer (μL)	2.5	2.5	2.5	2.5	2.5	2.5

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.3 Optimal RNP Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP concentration groups with final concentrations of 20, 60, 100, 140, 180, and 220 nM. Prepare the RNP systems according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.4 Optimal Mg²⁺ Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare six MgSO₄ groups with final concentrations of 5, 10, 25, 50, 75, and 100 mM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

6. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.1 Repeatability

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

Note: This experiment should be repeated multiple times by two or more operators during different time periods.

3.2 Sensitivity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare five concentration gradients, from 10¹ to 10⁵ copies/μL, of the simulated plasmid carrying SPN-lytA.

6. Add 2 μL of plasmid at the corresponding concentration gradient to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.3 Specificity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare the target simulated plasmid carrying SPN-lytA and the non-target simulated plasmids carrying HI-omp P6, GAS-speB, and BP-IS1663.

6. Add 2 μL of the corresponding type of simulated plasmid to the caps of the positive-reaction tubes and 2 μL of negative-control water to the caps of the negative-reaction tubes. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.4 Mechanism Interference Resistance

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare two groups of simulated plasmids carrying SPN-lytA. Dilute one group with a saliva-lysis-buffer-water solution and the other group with ddH₂O; dilute both groups to 10³ copies/μL.

6. Add 2 μL of the simulated plasmid carrying SPN-lytA to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator; prepare reverse primers R₁ and R₂ and forward primers F₁, F₂, F₃, and F₅; thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Divide the reactions into four groups, F₁R₁, F₂R₁, F₃R₁, and F₅R₂. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.1 Optimal Reaction Temperature

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument to six temperature gradients of 31, 34, 37, 40, 43, and 46 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.2 Optimal RNP Ratio

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP-ratio groups of 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Cas12a:gRNA	3:1	2:1	1.5:1	1:1.5	1:2	1:3
   Cas12a(μL)	1.875	1.667	1.5	1	0.833	0.625
   gRNA (μL)	0.625	0.833	1	1.5	1.667	1.875
   NE Buffer (μL)	5.5	5.5	5.5	5.5	5.5	5.5

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.3 Optimal RNP Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP concentration groups with final concentrations of 20, 60, 100, 140, 180, and 220 nM. Prepare the RNP systems according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.4 Optimal Mg²⁺ Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare six MgSO₄ groups with final concentrations of 5, 10, 25, 50, 75, and 100 mM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

6. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.1 Repeatability

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

Note: This experiment should be repeated multiple times by two or more operators during different time periods.

3.2 Sensitivity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare six concentration gradients, from 10⁰ to 10⁵ copies/μL, of the simulated plasmid carrying BP-IS1663.

6. Add 2 μL of the simulated plasmid carrying BP-IS1663 at the corresponding concentration gradient to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.3 Specificity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare the target simulated plasmid carrying BP-IS1663 and the non-target simulated plasmids carrying HI-omp P6, SPN-lytA, and GAS-speB.

6. Add 2 μL of the corresponding type of simulated plasmid to the caps of the positive-reaction tubes and 2 μL of negative-control water to the caps of the negative-reaction tubes. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.4 Mechanism Interference Resistance

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare two groups of simulated plasmids carrying BP-IS1663. Dilute one group with a saliva-lysis-buffer-water solution and the other group with ddH₂O; dilute both groups to 10³ copies/μL.

6. Add 2 μL of the simulated plasmid carrying BP-IS1663 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator; prepare reverse primers R₁, R_2, R₃, and R₄ and forward primer F₂; thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Divide the reactions into four groups, F₂R₁, F₂R₂, F₂R₃, and F₂R₄. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.1 Optimal Reaction Temperature

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument to six temperature gradients of 31, 34, 37, 40, 43, and 46 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.2 Optimal RNP Ratio

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP-ratio groups of 3:1, 2:1, 1.5:1, 1:1.5, 1:2, and 1:3. Prepare the RNP systems according to the reaction-system table below, and then incubate at 37 °C for 30 min.

   Cas12a:gRNA	3:1	2:1	1.5:1	1:1.5	1:2	1:3
   Cas12a(μL)	2.625	2.333	2.1	1.4	1.167	0.875
   gRNA(μL)	0.875	1.167	1.4	2.1	2.333	2.625
   NE Buffer (μL)	4.5	4.5	4.5	4.5	4.5	4.5

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.3 Optimal RNP Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare six RNP concentration groups with final concentrations of 20, 60, 100, 140, 180, and 220 nM. Prepare the RNP systems according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

2.4 Optimal Mg²⁺ Concentration

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. Prepare six MgSO₄ groups with final concentrations of 5, 10, 25, 50, 75, and 100 mM.

5. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

6. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.1 Repeatability

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

6. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

7. Collect and process the data.

Note: This experiment should be repeated multiple times by two or more operators during different time periods.

3.2 Sensitivity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare five concentration gradients, from 10¹ to 10⁵ copies/μL, of the simulated plasmid carrying HI-omp P6.

6. Add 2 μL of the simulated plasmid carrying HI-omp P6 at the corresponding concentration gradient to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.3 Specificity

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare the target simulated plasmid carrying HI-omp P6 and the non-target simulated plasmids carrying SPN-lytA, GAS-speB, and BP-IS1663.

6. Add 2 μL of the corresponding type of simulated plasmid to the caps of the positive-reaction tubes and 2 μL of negative-control water to the caps of the negative-reaction tubes. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.

3.4 Mechanism Interference Resistance

Experimental Procedure

1. Remove the reagents required for the experiment from the refrigerator, thaw, vortex, and centrifuge them, and set them aside for use.

2. Prepare the RNP system according to the reaction-system table, and then incubate at 37 °C for 30 min.

3. Prepare lyophilized bead tubes and prepare the RPA system in the lyophilized bead tubes according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture to the bottoms of the 8-tube PCR strips.

4. After incubation of the RNP system, prepare the CRISPR system according to the reaction-system table. Vortex and centrifuge, mix thoroughly, and aliquot the mixture onto the inner walls of the 8-tube PCR strips.

5. Prepare two groups of simulated plasmids carrying HI-omp P6. Dilute one group with a saliva-lysis-buffer-water solution and the other group with ddH₂O; dilute both groups to 10³ copies/μL.

6. Add 2 μL of the simulated plasmid carrying HI-omp P6 to the cap of each positive-reaction tube and 2 μL of negative-control water to the cap of each negative-reaction tube. Close the caps of the 8-tube PCR strips.

7. Set the PCR instrument parameters to 40 °C for 40 cycles. Centrifuge the 8-tube PCR strips to thoroughly mix all reagents, place them in the instrument reaction block, and start the reaction.

8. Collect and process the data.