Results

1. Design Overview

This section presents the experimental results from the following three stages in sequence, with a focus on the establishment and validation of the detection systems:

1. Primer screening: The amplification rate, endpoint fluorescence intensity, and background signal of different candidate primer combinations were compared to select the optimal RPA primer-crRNA combination for detection of each pathogen;

2. System optimization: Single-factor experiments were used to investigate key parameters, including the optimal reaction temperature, optimal molar ratio of RNP (Cas12a:crRNA), optimal RNP concentration, and optimal Mg²⁺ concentration, to determine the optimal reaction conditions for each detection system;

3. Performance evaluation: Under the optimized conditions, the repeatability, sensitivity, specificity, and resistance to matrix interference of each detection system were further evaluated.

Because the target characteristics and reaction properties differ among pathogens, primer-combination screening, condition optimization, and performance evaluation were conducted separately for five pathogens, successfully establishing highly sensitive and highly specific detection systems suitable for multiple pathogens.

2. Simulated Plasmid Carrying the RSV N Gene

2.1 Primer Screening

As shown in Figure 1, we selected the top three and the 10th of the top 10 RPA primer-crRNA combinations screened and ranked by the in silico DNABERT-6 model for the RSV N gene for wet-lab testing. The wet-lab results showed that all three top-ranked primer-crRNA combinations produced clear fluorescence signals in the positive-sample system, whereas fluorescence readings in the negative control group remained low, with no evident nonspecific activation. Among the candidate primer-crRNA combinations, combination 10 showed a later fluorescence peak in positive samples, limited signal amplification, and only a small signal difference between positive and negative samples; therefore, its overall performance was clearly inferior to that of the top three combinations. Notably, candidate combination 2 performed best in the wet-lab experiment, corresponding to F₁R₂. The result was consistent with the in silico ranking, and the optimal RPA primer-crRNA combination was therefore determined for subsequent use.

Figure 1 shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the RSV N gene. — **Figure 1** shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the RSV N gene.

2.2 System Optimization

2.2.1 Optimal Reaction Temperature

As shown in Figure 2, the optimal reaction temperature for the RPA-CRISPR cascade reaction system was 41 °C. This result indicates that 41 °C provides an appropriate balance between RPA amplification and Cas12a cleavage, thereby yielding the optimal fluorescence signal output.

Figure 2 shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system. — **Figure 2** shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system.

2.2.2 Optimal RNP Ratio (Cas12a:gRNA)

As shown in Figure 3, the optimal Cas12a:gRNA ratio in the RPA-CRISPR cascade reaction system was 3:1. The results indicate that, relative to gRNA, a moderately higher proportion of Cas12a facilitates efficient assembly of functional RNP complexes and enhances nuclease cleavage efficiency after target activation.

Figure 3 shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system. — **Figure 3** shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system.

2.2.3 Optimal RNP Concentration

As shown in Figure 4, the optimal final working concentration of the RNP complex in the RPA-CRISPR cascade reaction system was 25 nM. This result indicates that, within a certain range, an excessively high RNP concentration leads to a decrease in the fluorescence signal, which may be related to nonspecific interactions or reagent inhibition.

Figure 4 shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system. — **Figure 4** shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system.

2.2.4 Optimal Mg²⁺ Concentration

As shown in Figure 5, the optimal Mg²⁺ concentration for the RPA-CRISPR cascade reaction system was 20 mM. As a key enzymatic cofactor, Mg²⁺ at 20 mM can balance amplification and cleavage activities; deviation from this level inhibits overall reaction performance.

Figure 5 shows the fluorescence curves for optimization of the optimal Mg2+ concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system. — **Figure 5** shows the fluorescence curves for optimization of the optimal Mg²⁺ concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system.

2.2.5 Optimal Probe Concentration

As shown in Figure 6, the optimal probe concentration for the RPA-CRISPR cascade reaction system was 2000 nM. At 2000 nM, the probe concentration provides sufficient cleavage substrate for a strong signal while avoiding an increase in background caused by an excessive amount of probe.

Figure 6 shows the fluorescence curves for optimization of the optimal probe concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system. — **Figure 6** shows the fluorescence curves for optimization of the optimal probe concentration of the simulated plasmid carrying the RSV N gene in the RPA-CRISPR system.

2.3 Performance Evaluation

2.3.1 Repeatability Evaluation

Two operators performed parallel experiments at different time points. The individual plots in Figure 7 show six independent fluorescence kinetic curves obtained by the two operators on different dates and at different times. The rising trends of the positive-sample curves were highly similar, with only slight differences among the experimental groups, while all negative controls remained at baseline throughout the entire process, with no evident increase in fluorescence. The six sets of positive data were then combined to obtain the overlaid curves shown in Figure 8. The highly overlapping positive kinetic curves directly indicate good stability of the detection method. The endpoint fluorescence signals at 25 min from the six experiments were then averaged, giving a mean of 29688.90 and a standard deviation of 1534.98. The coefficient of variation (CV) of the endpoint signal was approximately 5.17%.

The visual consistency of the individual and overlaid plots, together with quantitative CV analysis, demonstrates that the RPA-CRISPR/Cas12a detection system has good inter-operator and inter-batch repeatability.

Figure 7 shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. — **Figure 7** shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene.

Figure 8 shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. — **Figure 8** shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene.

2.3.2 Sensitivity Evaluation

In the RPA-CRISPR cascade reaction system, the sensitivity for the simulated plasmid carrying the RSV N gene was 1.307 × 10²copies/µL, indicating that a positive signal could be successfully detected when the target nucleic acid concentration reached 130.7 copies/µL. The results are shown in Figure 9.

Figure 9 shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. — **Figure 9** shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene.

2.3.3 Specificity Evaluation

Specificity validation showed that the complete RPA-CRISPR/Cas12a detection system generated a positive fluorescence signal only for the target simulated plasmid. None of the tested simulated plasmids from non-target pathogens showed an evident increase in fluorescence, and their signal levels were essentially consistent with the negative control. These results indicate that the parameter-optimized detection system has excellent target-recognition specificity, can effectively distinguish the target pathogen from non-target pathogens, and meets the specificity requirements for actual sample testing. The results are shown in Figure 10.

Figure 10 shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. — **Figure 10** shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene.

2.3.4 Evaluation of Resistance to Matrix Interference

Matrix-interference testing showed that the overall fluorescence-increase trends were similar between the saliva-lysis-buffer matrix group and the pure-water control group, with only small differences in fluorescence for positive samples, and the positive signal in the saliva-containing group was slightly lower. Even in the presence of the complex saliva-lysis-buffer matrix, the RPA-CRISPR/Cas12a detection system consistently recognized the target simulated plasmid and clearly distinguished positive samples from negative controls; all negative controls remained at a stable baseline, with no nonspecific increase in fluorescence. These results indicate that the optimized system has good resistance to matrix interference. The complex saliva components and lysis buffer caused only slight inhibition and did not significantly affect detection performance, supporting subsequent application to testing of real clinical saliva samples. The results are shown in Figure 11.

Figure 11 shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. Positive and negative controls were tested under conditions with and without a saliva matrix. — **Figure 11** shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the RSV N gene. Positive and negative controls were tested under conditions with and without a saliva matrix.

Simulated Plasmid Carrying the hMPV-B N Gene

3.1 Primer Screening

As shown in Figure 12, we selected the top three and the 10th of the top 10 RPA primer-crRNA combinations screened and ranked by the in silico DNABERT-6 model for the hMPV-B N gene for experimental testing. The wet-lab results showed that all three top-ranked primer-crRNA combinations produced clear fluorescence signals in the positive-sample system; in contrast, fluorescence readings in the negative control group remained low, with no evident nonspecific activation. Candidate primer-crRNA combination 10 showed a later onset of fluorescence in positive samples, limited signal amplification, and only a small signal difference between positive and negative samples; therefore, its overall performance was clearly inferior to that of the top three combinations. Notably, candidate combination 1 performed best in the wet-lab experiment, corresponding to F₅R₅. The result was consistent with the in silico ranking, and F₅R₅ was therefore determined as the optimal RPA primer-crRNA combination.

Figure 12 shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the hMPV-B N gene. — **Figure 12** shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the hMPV-B N gene.

3.2 System Optimization

3.2.1 Optimal Reaction Temperature

As shown in Figure 13, the optimal reaction temperature for the RPA-CRISPR cascade reaction system was 42 °C. This result indicates that 42 °C provides an appropriate balance between RPA amplification and Cas12a cleavage, thereby yielding the optimal fluorescence signal output.

Figure 13 shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system. — **Figure 13** shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system.

3.2.2 Optimal RNP Ratio (Cas12a:gRNA)

As shown in Figure 14, the optimal Cas12a:gRNA ratio in the RPA-CRISPR cascade reaction system was 1:0.8. The results indicate that, relative to gRNA, a moderately higher proportion of Cas12a facilitates efficient assembly of functional RNP complexes and enhances nuclease cleavage efficiency after target activation.

Figure 14 shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system. — **Figure 14** shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system.

3.2.3 Optimal RNP Concentration

As shown in Figure 15, the optimal final working concentration of the RNP complex in the RPA-CRISPR cascade reaction system was 100 nM. This result indicates that, within a certain range, increasing the RNP concentration can improve signal output, whereas an excessively high RNP concentration leads to a decrease in the fluorescence signal, which may be related to nonspecific interactions or reagent inhibition.

Figure 15 shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system. — **Figure 15** shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system.

3.2.4 Optimal Mg²⁺ Concentration

As shown in Figure 16, the optimal Mg²⁺ concentration for the RPA-CRISPR cascade reaction system was 24 mM. As a key enzymatic cofactor, Mg²⁺ at 24 mM can balance amplification and cleavage activities; deviation from this level inhibits overall reaction performance.

Figure 16 shows the fluorescence curves for optimization of the optimal Mg2+ concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system. — **Figure 16** shows the fluorescence curves for optimization of the optimal Mg²⁺ concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system.

3.2.5 Optimal Probe Concentration

As shown in Figure 17, the optimal probe concentration for the RPA-CRISPR cascade reaction system was 2000 nM. At 2000 nM, the probe concentration provides sufficient cleavage substrate for a strong signal while avoiding an increase in background caused by an excessive amount of probe.

Figure 17 shows the fluorescence curves for optimization of the optimal probe concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system. — **Figure 17** shows the fluorescence curves for optimization of the optimal probe concentration of the simulated plasmid carrying the hMPV-B N gene in the RPA-CRISPR system.

3.3 Performance Evaluation

3.3.1 Repeatability Evaluation

Two operators performed parallel experiments at different time points. The individual plots in Figure 18 show six independent fluorescence kinetic curves obtained by the two operators on different dates and at different times. The rising trends of the positive-sample curves were highly similar; the negative controls remained at baseline throughout the entire process, with no evident increase in fluorescence, and only slight differences were observed among experimental batches. The six sets of positive data were then combined to obtain the overlaid curves shown in Figure 19. The highly overlapping positive kinetic curves directly reflect the stability of the system. The endpoint fluorescence signals at 25 min from the six experiments were then averaged, giving a mean of 1.09 × 10⁷ and a standard deviation of 7.44 × 10⁵. The coefficient of variation (CV) of the endpoint signal was approximately 6.83%.

The visual consistency of the individual and overlaid plots, together with quantitative CV analysis, demonstrates that the RPA-CRISPR/Cas12a detection system for the simulated plasmid carrying the hMPV-B N gene has good inter-operator and inter-batch repeatability.

Figure 18 shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene. — **Figure 18** shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene.

Figure 19 shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene. — **Figure 19** shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene.

3.3.2 Sensitivity Evaluation

In the RPA-CRISPR cascade reaction system, the sensitivity for the simulated plasmid carrying the hMPV-B N gene was 1.43 × 10¹ copies/µL, indicating that a detectable positive signal could be generated when the target nucleic acid concentration in the sample reached only 14.3 copies/µL. The results are shown in Figure 20.

Figure 20 shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene. — **Figure 20** shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene.

3.3.3 Specificity Evaluation

Specificity validation showed that the complete RPA-CRISPR/Cas12a detection system generated a positive fluorescence signal only for the simulated plasmid carrying the hMPV-B N gene. No evident increase in fluorescence was observed for any simulated plasmid from a non-target pathogen, and the signal levels were essentially consistent with the negative control. These results indicate that the parameter-optimized detection system has excellent target-recognition specificity, can effectively distinguish the target pathogen from other non-target pathogens, and meets the specificity requirements for actual sample testing. The results are shown in Figure 21.

Figure 21 shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene. — **Figure 21** shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene.

3.3.4 Evaluation of Resistance to Matrix Interference

As shown in Figure 22, matrix-interference testing showed that the overall fluorescence-increase trends were similar between the saliva-lysis-buffer matrix group and the pure-water control group, with only small differences in fluorescence for positive samples, and the positive signal in the saliva-containing group was slightly lower. Even in the presence of the complex saliva-lysis-buffer matrix, the RPA-CRISPR/Cas12a detection system consistently recognized the simulated plasmid carrying the hMPV-B N gene and clearly distinguished positive samples from negative controls; all negative controls remained at a stable baseline, with no nonspecific increase in fluorescence. These results indicate that the optimized system has good resistance to matrix interference. The complex saliva components and lysis buffer caused only slight inhibition and did not significantly affect detection performance, supporting subsequent application to testing of real clinical saliva samples.

Figure 22 shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene. — **Figure 22** shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the hMPV-B N gene.

Simulated Plasmid Carrying the SPN-lytA Gene

4.1 Primer Screening

As shown in Figure 23, we selected the top three and the 10th of the top 10 candidate RPA primer-crRNA combinations screened and ranked by the in silico DNABERT-6 model for the SPN-lytA gene for wet-lab testing. The wet-lab results showed that all three top-ranked primer-crRNA combinations produced clear fluorescence signals in the positive-sample system, whereas fluorescence readings in the negative control group remained low, indicating no evident nonspecific activation. Among the candidate primer-crRNA combinations, combination 10 showed a later onset of fluorescence in positive samples, limited signal amplification, and only a small signal difference between positive and negative samples; therefore, its overall performance was clearly inferior to that of the top three combinations. Notably, candidate combination 3 performed best in the wet-lab experiment, corresponding to F₂R₃. The result was consistent with the in silico ranking, and the optimal RPA primer-crRNA combination was therefore determined.

Figure 23 shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the SPN-lytA gene. — **Figure 23** shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the SPN-lytA gene.

4.2 System Optimization

4.2.1 Optimal Reaction Temperature

As shown in Figure 24, the optimal reaction temperature for the RPA-CRISPR cascade reaction system was 40 °C. This result indicates that 40 °C provides an appropriate balance between RPA amplification and Cas12a cleavage, thereby yielding the optimal fluorescence signal output.

Figure 24 shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system. — **Figure 24** shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system.

4.2.2 Optimal RNP Ratio (Cas12a:gRNA)

As shown in Figure 25, the optimal Cas12a:gRNA ratio in the RPA-CRISPR cascade reaction system was 3:1. The results indicate that, relative to gRNA, a moderately higher proportion of Cas12a facilitates efficient assembly of functional RNP complexes and enhances nuclease cleavage efficiency after target activation.

Figure 25 shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system. — **Figure 25** shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system.

4.2.3 Optimal RNP Concentration

As shown in Figure 26, the optimal final working concentration of the RNP complex in the RPA-CRISPR cascade reaction system was 220 nM. This result indicates that, within a certain range, increasing the RNP concentration can improve signal output, whereas an excessively high RNP concentration leads to a decrease in the fluorescence signal, which may be related to nonspecific interactions or reagent inhibition.

Figure 26 shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system. — **Figure 26** shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system.

4.2.4 Optimal Mg²⁺ Concentration

As shown in Figure 27, the optimal Mg²⁺ concentration for the RPA-CRISPR cascade reaction system was 75 mM. As a key enzymatic cofactor, Mg²⁺ at 75 mM can balance amplification and cleavage activities; deviation from this level inhibits overall reaction performance.

Figure 27 shows the fluorescence curves for optimization of the optimal Mg2+ concentration of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system. — **Figure 27** shows the fluorescence curves for optimization of the optimal Mg²⁺ concentration of the simulated plasmid carrying the SPN-lytA gene in the RPA-CRISPR system.

4.3 Performance Evaluation

4.3.1 Repeatability Evaluation

Two operators performed parallel experiments at different time points. The individual plots in Figure 28 show six independent fluorescence kinetic curves obtained by the two operators on different dates and at different times. The rising trends of the positive-sample curves were highly similar; the negative controls remained at baseline throughout the entire process, with no evident increase in fluorescence, and only slight differences were observed among experimental batches. The six sets of positive data were then combined to obtain the overlaid curves shown in Figure 29. The highly overlapping positive kinetic curves directly reflect the stability of the system. The endpoint fluorescence signals at 25 min from the six experiments were then averaged, giving a mean of 24711.87 and a standard deviation of 1837.92. The coefficient of variation (CV) of the endpoint signal was approximately 7.44%.

The visual consistency of the individual and overlaid plots, together with quantitative CV analysis, demonstrates that the RPA-CRISPR/Cas12a detection system for the simulated plasmid carrying the SPN-lytA gene has good inter-operator and inter-batch repeatability.

Figure 28 shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene. — **Figure 28** shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene.

Figure 29 shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene. — **Figure 29** shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene.

4.3.2 Sensitivity Evaluation

In the RPA-CRISPR cascade reaction system, the sensitivity for the simulated plasmid carrying the SPN-lytA gene was 1.0 × 10² copies/µL, indicating that a detectable positive signal could be generated when the target nucleic acid concentration in the sample reached only 100 copies/µL. The results are shown in Figure 30.

Figure 30 shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene. — **Figure 30** shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene.

4.3.3 Specificity Evaluation

Specificity validation showed that the complete RPA-CRISPR/Cas12a detection system generated a positive fluorescence signal only for the simulated plasmid carrying the SPN-lytA gene. No evident increase in fluorescence was observed for any simulated plasmid from a non-target pathogen, and the signal levels were essentially consistent with the negative control. These results indicate that the parameter-optimized detection system has excellent target-recognition specificity, can effectively distinguish the target pathogen from other non-target pathogens, and meets the specificity requirements for actual sample testing. The results are shown in Figure 31.

Figure 31 shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene. — **Figure 31** shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene.

4.3.4 Evaluation of Resistance to Matrix Interference

As shown in Figure 32, matrix-interference testing showed that the overall fluorescence-increase trends were similar between the saliva-lysis-buffer matrix group and the pure-water control group, with only small differences in fluorescence for positive samples. Even in the presence of the complex saliva-lysis-buffer matrix, the RPA-CRISPR/Cas12a detection system consistently recognized the simulated plasmid carrying the SPN-lytA gene and clearly distinguished positive samples from negative controls; all negative controls remained at a stable baseline, with no nonspecific increase in fluorescence. These results indicate that the optimized system has good resistance to matrix interference. The complex saliva components and lysis buffer caused only slight inhibition and did not significantly affect detection performance, supporting subsequent application to testing of real clinical saliva samples.

Figure 32 shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene. — **Figure 32** shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the SPN-lytA gene.

Simulated Plasmid Carrying the BP-IS1663 Gene

5.1 Primer Screening

As shown in Figure 33, we selected the top three and the 10th of the top 10 candidate RPA primer-crRNA combinations screened and ranked by the in silico DNABERT-6 model for the BP-IS1663 gene for wet-lab testing. The wet-lab results showed that all three top-ranked primer-crRNA combinations produced clear fluorescence signals in the positive-sample system, whereas fluorescence readings in the negative control group remained low, with no evident nonspecific activation. Among the candidate primer-crRNA combinations, combination 10 showed a later fluorescence peak in positive samples, limited signal amplification, and only a small signal difference between positive and negative samples; therefore, its overall performance was clearly inferior to that of the top three combinations. Notably, candidate combination 2 performed best in the wet-lab experiment, corresponding to F₅R₂. The result was consistent with the in silico ranking, and the optimal RPA primer-crRNA combination was therefore determined.

Figure 33 shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the BP-IS1663 gene. — **Figure 33** shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the BP-IS1663 gene.

5.2 System Optimization

5.2.1 Optimal Reaction Temperature

As shown in Figure 34, the optimal reaction temperature for the RPA-CRISPR cascade reaction system was 40 °C. This result indicates that 40 °C provides an appropriate balance between RPA amplification and Cas12a cleavage, thereby yielding the optimal fluorescence signal output.

Figure 34 shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system. — **Figure 34** shows the fluorescence curves for optimization of the optimal reaction temperature of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system.

5.2.2 Optimal RNP Ratio (Cas12a:gRNA)

As shown in Figure 35, the optimal Cas12a:gRNA ratio in the RPA-CRISPR cascade reaction system was 2:1. The results indicate that, relative to gRNA, a moderately higher proportion of Cas12a facilitates efficient assembly of functional RNP complexes and enhances nuclease cleavage efficiency after target activation.

Figure 35 shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system. — **Figure 35** shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system.

5.2.3 Optimal RNP Concentration

As shown in Figure 36, the optimal final working concentration of the RNP complex in the RPA-CRISPR cascade reaction system was 100 nM. This result indicates that, within a certain range, increasing the RNP concentration can improve signal output, whereas an excessively high RNP concentration leads to a decrease in the fluorescence signal, which may be related to nonspecific interactions or reagent inhibition.

Figure 36 shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system. — **Figure 36** shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system.

5.2.4 Optimal Mg²⁺ Concentration

As shown in Figure 37, the optimal Mg²⁺ concentration for the RPA-CRISPR cascade reaction system was 50 mM. As a key enzymatic cofactor, Mg²⁺ at 50 mM can balance amplification and cleavage activities; deviation from this level inhibits overall reaction performance.

Figure 37 shows the fluorescence curves for optimization of the optimal Mg2+ concentration of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system. — **Figure 37** shows the fluorescence curves for optimization of the optimal Mg²⁺ concentration of the simulated plasmid carrying the BP-IS1663 gene in the RPA-CRISPR system.

5.3 Performance Evaluation

5.3.1 Repeatability Evaluation

Two operators performed parallel experiments at different time points. The individual plots in Figure 38 show six independent fluorescence kinetic curves obtained by the two operators on different dates and at different times. The rising trends of the positive-sample curves were highly similar; the negative controls remained at baseline throughout the entire process, with no evident increase in fluorescence, and only slight differences were observed among experimental batches. The six sets of positive data were then combined to obtain the overlaid curves shown in Figure 39. The highly overlapping positive kinetic curves directly reflect the stability of the system. The endpoint fluorescence signals at 25 min from the six experiments were then averaged, giving a mean of 42439.09 and a standard deviation of 5546.89. The coefficient of variation (CV) of the endpoint signal was approximately 13.07%.

The visual consistency of the individual and overlaid plots, together with quantitative CV analysis, demonstrates that the RPA-CRISPR/Cas12a detection system for the simulated plasmid carrying the BP-IS1663 gene has good inter-operator and inter-batch repeatability.

Figure 38 shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene. — **Figure 38** shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene.

Figure 39 shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene. — **Figure 39** shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene.

5.3.2 Sensitivity Evaluation

In the RPA-CRISPR cascade reaction system, the sensitivity for the simulated plasmid carrying the BP-IS1663 gene was 1.0 × 10¹ copies/µL, indicating that a detectable positive signal could be generated when the target nucleic acid concentration in the sample reached only 10 copies/µL. The results are shown in Figure 40.

Figure 40 shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene. — **Figure 40** shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene.

5.3.3 Specificity Evaluation

Specificity validation showed that the complete RPA-CRISPR/Cas12a detection system generated a positive fluorescence signal only for the simulated plasmid carrying the BP-IS1663 gene. No evident increase in fluorescence was observed for any simulated plasmid from a non-target pathogen, and the signal levels were essentially consistent with the negative control. These results indicate that the parameter-optimized detection system has excellent target-recognition specificity, can effectively distinguish the target pathogen from other non-target pathogens, and meets the specificity requirements for actual sample testing. The results are shown in Figure 41.

Figure 41 shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene. — **Figure 41** shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene.

5.3.4 Evaluation of Resistance to Matrix Interference

As shown in Figure 42, matrix-interference testing showed that the overall fluorescence-increase trends were similar between the saliva-lysis-buffer matrix group and the pure-water control group, with only small differences in fluorescence for positive samples. Even in the presence of the complex saliva-lysis-buffer matrix, the RPA-CRISPR/Cas12a detection system consistently recognized the simulated plasmid carrying the BP-IS1663 gene and clearly distinguished positive samples from negative controls; all negative controls remained at a stable baseline, with no nonspecific increase in fluorescence. These results indicate that the optimized system has good resistance to matrix interference. The complex saliva components and lysis buffer caused only slight inhibition and did not significantly affect detection performance, supporting subsequent application to testing of real clinical saliva samples.

Figure 42 shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene. — **Figure 42** shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the BP-IS1663 gene.

Simulated Plasmid Carrying the HI-ompP6 Gene

6.1 Primer Screening

As shown in Figure 43, we selected the top three and the 10th of the top 10 RPA primer-crRNA combinations screened and ranked by the in silico DNABERT-6 model for the HI-ompP6 gene for wet-lab testing. The wet-lab results showed that all three top-ranked primer-crRNA combinations produced clear fluorescence signals in the positive-sample system, whereas fluorescence readings in the negative control group remained low, with no evident nonspecific activation. In contrast, the candidate primer-crRNA combination ranked 10th showed a later onset of fluorescence in positive samples, limited signal amplification, and only a small signal difference between positive and negative samples; therefore, its overall performance was clearly inferior to that of the top three combinations. Notably, candidate combination 1 performed best in the wet-lab experiment, corresponding to F₂R₂. The result was consistent with the in silico ranking, and the optimal RPA primer-crRNA combination was therefore determined.

Figure 43 shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the HI-ompP6 gene. — **Figure 43** shows the fluorescence curves used to optimize candidate primer-crRNA combinations targeting the HI-ompP6 gene.

6.2 System Optimization

6.2.1 Optimal Reaction Temperature

As shown in Figure 44, the optimal reaction temperature for the RPA-CRISPR cascade reaction system was 40 °C. This result indicates that 40 °C provides an appropriate balance between RPA amplification and Cas12a cleavage, thereby yielding the optimal fluorescence signal output.

Figure 44 shows the fluorescence curves for the optimal reaction temperature of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system. — **Figure 44** shows the fluorescence curves for the optimal reaction temperature of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system.

6.2.2 Optimal RNP Ratio (Cas12a:gRNA)

As shown in Figure 45, the optimal Cas12a:gRNA ratio in the RPA-CRISPR cascade reaction system was 3:1. The results indicate that, relative to gRNA, a moderately higher proportion of Cas12a facilitates efficient assembly of functional RNP complexes and enhances nuclease cleavage efficiency after target activation.

Figure 45 shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system. — **Figure 45** shows the fluorescence curves for optimization of the optimal RNP ratio of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system.

6.2.3 Optimal RNP Concentration

As shown in Figure 46, the optimal final working concentration of the RNP complex in the RPA-CRISPR cascade reaction system was 140 nM. This result indicates that, within a certain range, increasing the RNP concentration can improve signal output, whereas an excessively high RNP concentration leads to a decrease in the fluorescence signal, which may be related to nonspecific interactions or reagent inhibition.

Figure 46 shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system. — **Figure 46** shows the fluorescence curves for optimization of the optimal RNP concentration of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system.

6.2.4 Optimal Mg²⁺ Concentration

As shown in Figure 47, the optimal Mg²⁺ concentration for the RPA-CRISPR cascade reaction system was 75 mM. As a key enzymatic cofactor, Mg²⁺ at 75 mM can balance amplification and cleavage activities; deviation from this level inhibits overall reaction performance.

Figure 47 shows the fluorescence curves for optimization of the optimal Mg2+ concentration of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system. — **Figure 47** shows the fluorescence curves for optimization of the optimal Mg²⁺ concentration of the simulated plasmid carrying the HI-ompP6 gene in the RPA-CRISPR system.

6.3 Performance Evaluation

6.3.1 Repeatability Evaluation

Two operators performed parallel experiments at different time points. The individual plots in Figure 48 show six independent fluorescence kinetic curves obtained by the two operators on different dates and at different times. The rising trends of the positive-sample curves were highly similar; the negative controls remained at baseline throughout the entire process, with no evident increase in fluorescence, and only slight differences were observed among experimental batches. The six sets of positive data were then combined to obtain the overlaid curves shown in Figure 49. The highly overlapping positive kinetic curves directly reflect the stability of the system. The endpoint fluorescence signals at 25 min from the six experiments were then averaged, giving a mean of 12275.88 and a standard deviation of 2548.85. The coefficient of variation (CV) of the endpoint signal was approximately 20.76%.

The visual consistency of the individual and overlaid plots, together with quantitative CV analysis, demonstrates that the RPA-CRISPR/Cas12a detection system for the simulated plasmid carrying the HI-ompP6 gene has good inter-operator and inter-batch repeatability.

Figure 48 shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene. — **Figure 48** shows the fluorescence curves used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene.

Figure 49 shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene. — **Figure 49** shows the overlaid fluorescence curves from six parallel experiments used to evaluate the repeatability of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene.

6.3.2 Sensitivity Evaluation

In the RPA-CRISPR cascade reaction system, the sensitivity for the simulated plasmid carrying the HI-ompP6 gene was 1.0 × 10³ copies/µL, indicating that a detectable positive signal could be generated when the target nucleic acid concentration in the sample reached only 1000 copies/µL. The results are shown in Figure 50.

Figure 50 shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene. — **Figure 50** shows the fluorescence curves used to evaluate the sensitivity of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene.

6.3.3 Specificity Evaluation

Specificity validation showed that the complete RPA-CRISPR/Cas12a detection system generated a positive fluorescence signal only for the simulated plasmid carrying the HI-ompP6 gene. No evident increase in fluorescence was observed for any simulated plasmid from a non-target pathogen, and the signal levels were essentially consistent with the negative control. These results indicate that the parameter-optimized detection system has excellent target-recognition specificity, can effectively distinguish the target pathogen from other non-target pathogens, and meets the specificity requirements for actual sample testing. The results are shown in Figure 51.

Figure 51 shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene. — **Figure 51** shows the fluorescence curves used to evaluate the specificity of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene.

6.3.4 Evaluation of Resistance to Matrix Interference

As shown in Figure 52, matrix-interference testing showed that the overall fluorescence-increase trends were similar between the saliva-lysis-buffer matrix group and the pure-water control group, with only small differences in fluorescence for positive samples. Even in the presence of the complex saliva-lysis-buffer matrix, the RPA-CRISPR/Cas12a detection system consistently recognized the simulated plasmid carrying the HI-ompP6 gene and clearly distinguished positive samples from negative controls; all negative controls remained at a stable baseline, with no nonspecific increase in fluorescence. These results indicate that the optimized system has good resistance to matrix interference. The complex saliva components and lysis buffer caused only slight inhibition and did not significantly affect detection performance, supporting subsequent application to testing of real clinical saliva samples.

Figure 52 shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene. — **Figure 52** shows the fluorescence curves used to evaluate resistance to matrix interference of the RPA-CRISPR system for the simulated plasmid carrying the HI-ompP6 gene.