Genomic (DNA or cDNA) of M. hyopneumoniae, Mycoplasma hyorhinis (M. hyorhinis), A. pleuropneumoniae, H. parasuis, S. suis, P. multocida, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), Mycoplasma capricolum (M. capricolum), Mycoplasma synoviae (M. synoviae) and Mycoplasma gallisepticum (M. gallisepticum) were stored in the State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In addition, we gratefully acknowledge the contribution of 51 lung tissue samples and 25 nasal swab samples from pigs, kindly provided by other laboratories at the Harbin Veterinary Research Institute. The Animal Ethics Committee of Harbin Veterinary Research Institute did not require ethical review or approval for this study.

The P36 gene of M. hyopneumoniae has been shown to be highly homologous (Caron et al., 2000), so we selected it as a test gene and used fragments of the conserved region for the design of crRNA. Four DNA templates for crRNA synthesis were designed using the CHOPCHOP online tool (Montague et al., 2014). The four DNA templates were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The four DNA templates underwent an annealing heat treatment. Subsequently, in vitro transcription was performed by incubating at 37°C for 16 hours, following the instructions provided by the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs, Beverly, MA, USA). The final transcription products were treated with DNase I (New England Biolabs) to remove residual DNA template and purified using the Monarch RNA Cleanup Kit Protocol Card (New England Biolabs). The concentration of the final crRNA was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -80°C for future use. The sequences of all oligonucleotides synthesized in this study are presented in Table 1 .

Plasmid was constructed by PCR amplification of genomic DNA using the primer pairs Mhp P36 F1 and R1 ( Table 1 ) designed in the conserved regions of the P36 gene. The PCR products were purified using a gel extraction kit (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. The purified fragments were ligated into the pMD-18T vector (Takara Bio Inc., Dalian, China) and transformed into DH5α competent cells (Takara Bio Inc.) for overnight culture, resulting in plasmid isolation. The concentration of the plasmid was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and the standard plasmid copy number was calculated using the following formula: copies/μL = (A260 (ng/μL) × 10⁻⁹ × 6.02 × 10²³)/(DNA length × 650). A ten-fold dilution series ranging from 1 × 10⁸ to 1 × 10^-1 copies/μL of the standard plasmid was prepared using EASY Dilution (Takara Bio Inc.) and stored at -20°C for subsequent experiments.

Standard RAA reactions were conducted according to the instructions of the Basic Nucleic Acid Amplification Kit (ZC Bio-Sci&Tech Co. Ltd, Hangzhou, China). Each reaction mixture contained 25 µL of A Buffer, 2 µL each of forward and reverse primers (10 µM), 5 µL of genomic DNA, 2.5 µL of B buffer, and 13.5 µL of nuclease-free H₂O. The mixture was incubated in a water bath at 39°C for 30 min. Six primer pairs, denoted as RAA-F1/R1, RAA-F2/R2, RAA-F3/R3, RAA-F4/R3, RAA-F4/R4, and RAA-F4/R2 ( Table 1 ), were generated using Primer 5.0 software. These primers were designed based on sequences flanking the crRNA probe region, ensuring no overlap with the crRNA region. The design parameters for these primers include a product length range of 150-250 bp, primer length between 30 and 35 bp, and GC content ranging from 30% to 70%.

Six primer pairs (RAA-F1/R1, RAA-F2/R2, RAA-F3/R3, RAA-F4/R3, RAA-F4/R4 and RAA-F4/R2) ( Table 1 ) were used to perform RAA reactions separately serving standard plasmids of 1 × 10³ copies/µL as a template. Primer pairs with higher amplification efficiency and specificity were conceived as candidates. Then, the amplification template was switched from standard plasmid to genome, and following the above procedure, the primer pair with the highest specificity and amplification efficiency among the candidate primer pairs was selected for subsequent experiments. Furthermore, we explored the optimal RAA amplification time. Reaction times of 15 min, 20 min, 25 min and 30 min were tested, with each experiment repeated three times. By comprehensively analyzing the fluorescence generation time, fluorescence curve trends, and fluorescence intensity influenced by the five different RAA reaction times, the optimal RAA reaction time was determined.

The unoptimized reaction system (50 μL) consisted of the following mixture: 1 μL LbCas12a (5 µM) (Bio-lifesci Co., Ltd., Guangzhou, China), 5 μL 10× buffer, 2 μL crRNA (5 µM) (in vitro synthesis), 2 μL ssDNA reporter probe (10 µM) (Sangon Biotech Co., Ltd., Shanghai, China), 3 μL template, with the remainder made up with nuclease-free H₂O. The mixture was incubated for 25 min at 37°C in a water bath and a microplate reader was used for detection (PerkinElmer, Waltham, MA, USA). The crRNA was screened using this reaction system and conditions and the most suitable crRNA was identified based on the final fluorescence intensity and reaction efficiency for subsequent experiments. In the assay, the final concentrations of LbCas12a, crRNA and ssDNA were optimized using the checkerboard method to optimize concentrations of LbCas12a and crRNA by fixing other components, which was set up with 30, 60, 120, 180 and 240 nM of crRNA and 20, 40, 60, 80, 100 and 200 nM of LbCas12a. ssDNA was optimized by fixing other components and adjusting the concentration to 100, 200, 300, 400 and 500 nM. Experimental data measured using a microplate reader (PerkinElmer) were processed to determine the optimal concentrations of LbCas12a, crRNA and ssDNA for the final reaction system.

The results can be expressed in terms of fluorescence using a microplate reader and visualized under blue light or through lateral flow assay (LFA). For fluorescence detection, the ssDNA reporter is labeled with FAM and BHQ1 in the fluorescent and quencher groups, respectively. We use a microplate reader (PerkinElmer) with excitation and emission wavelengths set at 494 nm and 518 nm to measure the fluorescence intensity. Additionally, a blue light transilluminator can be used to observe fluorescence, where the presence of green fluorescence serves as an indicator of the target DNA’s existence. For LFA detection, the ssDNA reporter is labeled with FAM and biotin at the 5’ and 3’ ends, respectively. Following the instructions, we place the lateral flow assay strip (GenDx Biotech Co., Ltd., Suzhou, China) into the incubation product, yielding results within 2 min. The lateral flow strip comprises three distinct sections: the sample zone, the control line and the test line. The sample zone is coated with gold-labeled anti-FAM antibodies. The control line area is covered with streptavidin, which captures uncleaved ssDNA reporter molecules labeled with biotin. The test line area is coated with anti-mouse antibodies designed to detect the cleaved ssDNA reporter labeled with gold nanoparticles. For negative samples, the gold-labeled anti-biotin antibodies fully bind to the abundant biotin-labeled ssDNA reporter, resulting in complexes that are intercepted by anti-FAM antibodies at the control line. Conversely, for positive samples, the ssDNA reporter is cleaved, leading to the accumulation of gold-labeled anti-biotin antibody at the test line. The colorimetric process should ideally be completed within 4 min. Positive results are indicated by the presence of a red control line and test line, or only a red test line. In contrast, results are considered negative when only the control line shows a red color, and invalid when no lines are displayed.

In order to investigate the specificity of the RAA-CRISPR/Cas12a fluorescence detection method, genome DNA of M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, H. parasuis, S. suis, P. multocida, PCV2, PRV, M. capricolum, M. synoviae and M. gallisepticum and genome cDNA of PRRSV and SIV were detected by the final reaction system. Meanwhile, nuclease-free H₂O was used as a negative control.

To evaluate the sensitivity of the RAA-CRISPR/Cas12a fluorescence detection method, 10-fold dilutions of standard plasmids ranging from 1 × 10⁴ to 1 × 10^-1 copies/μL and 5 copies/μL of standard plasmid were used as templates to determine the limit of detection (LoD) for the RAA-CRISPR/Cas12a-fluorescence. Additionally, using the 10-fold dilution of 1 × 10⁸–1 × 10^-1 copies/μL of the standard plasmid as templates, we tested the sensitivity of qPCR according to the recommended Chinese entry–exit inspection and quarantine industry standard (SN/T4104-2015). The qPCR reaction mixture (20 μL) contained 10 μL Premix ExTaq (probe qPCR) (2×), 0.4 μL of each Mhp-SN-F/R primer (10 µM), 0.8 μL Mhp-SN-probe (10 µM), 0.4 μL Rox Reference DyeII, 5 μL template and 3 μL nuclease-free H₂O. The cycling conditions were set as follows: initial denaturation at 95°C for 3 min, subsequently proceeding to 40 cycles, each consisting of denaturation at 95°C for 15 seconds and annealing at 55°C for 45 seconds.

The reaction time of the RAA-CRISPR/Cas12a lateral flow assay was optimized for use in the final reaction system. Four sets with reaction times of 5 min, 10 min, 15 min and 20 min were optimized using 1 × 10² copies/µL of standard plasmid as templates, and incubated in a 37°C water bath to determine the minimum reaction time. The optimal reaction time was selected by observing the test line and control line.

In order to evaluate the specificity of the RAA-CRISPR/Cas12a LFA, the thirteen pathogens M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, H. parasuis, S. suis, P. multocida, PRRSV, SIV, PCV2, PRV, M. capricolum, M. synoviae and M. gallisepticum were analyzed. Nuclease-free H₂O was used as a negative control.

For sensitivity assessment, 10-fold serial dilutions of 1 × 10⁴–1 × 10^-1 copies/μL and 5 copies/μL of the standard plasmid were used as templates to determine the LoD for the RAA-CRISPR/Cas12a LFA.

Specificity and sensitivity of M. hyopneumoniae were determined by observing the control and detection lines of the test strips.

Genomic DNA was extracted from 51 lung tissue samples (17 apical lobes, 17 cardiac lobes and 17 diaphragmatic lobes) using a blood/cell/tissue genomic DNA extraction kit (Tiangen Biotech Co. Ltd., Beijing, China) and from 25 nasal swab samples using a bacterial genomic DNA extraction kit (Tiangen Biotech Co. Ltd.). The DNA samples were tested using the final RAA-CRISPR/Cas12a-fluorescence and RAA-CRISPR/Cas12a LFA. To validate the clinical performance, the same samples were analyzed by PCR recommended by the Chinese national standard (GB/T 35909-2018) and qPCR recommended by the Chinese entry–exit inspection and quarantine industry standard (SN/T4104-2015). Genomic DNA of M. hyopneumoniae and nuclease-free H₂O were used as positive and negative controls, respectively. The reaction mixture (25 μL) for PCR contained 12.5 μL 2× Taq PCR StarMix (Dye), 2 μL of each Mhp-GB-F/R primer (10 µM), 3 μL template and 5.5 μL nuclease-free H₂O. The reaction conditions were set as follows: pre-denaturation at 94°C for 2 min, followed by PCR performed for 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.

Data analysis and graphic design were processed by GraphPad Prism v.8.1.2 software. Data in the figures are presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used for multigroup comparisons. All comparisons were considered statistically significant at P < 0.05 and highly significant at P < 0.001.

An overview of the rapid detection platform for M. hyopneumoniae based on RAA-CRISPR/Cas12a is depicted in Figure 1 . First, genomic DNA is extracted from bacterial culture using a bacterial genomic DNA extraction kit. Next, the target DNA is amplified through RAA at 39°C, followed by transfer of the RAA products into the CRISPR/Cas12a reaction system. Finally, the presence of M. hyopneumoniae is determined by visual inspection under blue light, utilization of a lateral flow assay, or fluorescence measured using a microplate reader. The entire process can be completed within one hour.

Because the primer sequences are important in RAA amplification, primer screening is necessary. We used standard plasmid and DNA of M. hyopneumoniae as templates to identify the optimal primer pair. Four primer pairs (RAA-F1/R1, RAA-F2/R2, RAA-F4/R4, RAA-F4/R2) were selected using a standard plasmid of 1 × 10³ copies/µL as a template based on the amplification profile ( Figure 2A ). Then, RAA-F1/R1 was selected for subsequent experiments, as it exhibited the most efficient performance as well as being highly specific when tested using DNA of M. hyopneumoniae as a template ( Figure 2B ). Based on the five sets of reaction times designed for the RAA, the optimal RAA reaction time was determined to be 15 min by considering the fluorescence intensity and background fluorescence values influenced by different reaction durations ( Figure 2C , P < 0.001).

Four designed crRNA probes were capable of recognizing distinct 24-nucleotide regions of the P36 gene ( Figure 3A ). Among them, crRNA 1 exhibited the highest fluorescence value ( Figure 3B ) and the fastest reaction rate. Therefore, crRNA 1 was selected for use in subsequent experiments. The final optimized reaction system comprised 0.6 μL LbCas12a (final concentration of 60 nM) ( Figure 3D ), 5 μL 10 × buffer, 1.8 μL crRNA (final concentration of 180 nM) ( Figure 3D ), 2 μL ssDNA reporter probe (final concentration of 400 nM) ( Figure 3C ), and 3 μL RAA product, with the volume made up to 50 μL with nuclease-free H₂O.

Genomic DNA of M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, H. parasuis, S. suis, P. multocida, PCV2, PRV, M. capricolum, M. synoviae and M. gallisepticum and genomic cDNA of PRRSV and SIV as templates were tested to evaluate the specificity of the RAA-CRISPR/Cas12a-fluorescence system. Only DNA of M. hyopneumoniae reacted rapidly with high fluorescence intensity (P < 0.001) and did not cross-react with other pathogens ( Figure 4A ). The results showed that the RAA-CRISPR/Cas12a fluorescence detection system was highly specific.

A 10-fold serial dilution series and 5 copies/μL of standard plasmids were used as templates for the RAA-CRISPR/Cas12a-fluorescence. The result showed that the LoD of the fluorescence assay was 1 copy/μL for M. hyopneumoniae ( Figure 4B , P < 0.001). At the same, the qPCR recommended by entry–exit inspection and quarantine industry standard (SN/T4104-2015) detected 1 × 10² copies/μL (CT < 36) ( Figure 4C ). This result indicates that the fluorescence assay is highly sensitive, being 100-fold more sensitive than qPCR.

The reaction time of the RAA-CRISPR/Cas12a lateral flow assay was optimized based on the final reaction system. When 1 × 10² copies/µL of standard plasmid was used as a template, the lateral flow strip only displayed the detection line after incubation for 15 min and 20 min ( Figure 5 ), indicating that it takes a minimum of 15 min for the reaction to be complete. Thus the shortest available reaction time for the RAA-CRISPR/Cas12a LFA was 15 min.

The specificity of the RAA-CRISPR/Cas12a LFA was evaluated for the thirteen pathogens M. hyopneumoniae, M. hyorhinis, A. pleuropneumoniae, H. parasuis, S. suis, P. multocida, PRRSV, SIV, PCV2, PRV, M. capricolum, M. synoviae and M. gallisepticum based on the final reaction system. In the LFA strips the test line was only visible with M. hyopneumoniae, for the others only the control line appeared ( Figure 6A ). The sensitivity of the RAA-CRISPR/Cas12a LFA was analyzed using a 10-fold serial dilution and 5 copies/μL of standard plasmids. The LoD was 5 copies/μL ( Figure 6B ).

In order to evaluate the performance of the established detection platform, 51 lung tissues samples and 25 nasal swab samples were tested using the final RAA-CRISPR/Cas12a fluorescence/LFA ( Figure 7 ). The positive rates for 51 lung tissue samples and 25 nasal swab samples were 100% (51/51) and 28% (7/25) in RAA-CRISPR/Cas12a fluorescence ( Table 2 , Figure 7 , P < 0.05). The same results were obtained by RAA-CRISPR/Cas12a LFA ( Table 2 , Figure 7 ). To verify the accuracy of the method, we tested the clinical samples using PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry–exit inspection and quarantine industry standard (SN/T4104-2015). The PCR results indicated a positive rate of 96% (49/51) among 51 lung tissue samples, and the nasal swab samples exhibited a 0% (0/25) positive rate among 25 samples ( Table 2 ). Additionally, the 51 lung tissue samples tested by qPCR were 100% (51/51) positive, meanwhile, the nasal swab samples showed a 16% (4/25) positive rate among the 25 samples examined ( Table 2 ). The results obtained using the RAA-CRISPR/Cas12a fluorescence/LFA method were consistent with those obtained from PCR (GB/T 35909-2018) and qPCR (SN/T4104-2015). This suggests that the novel approach is not only accurate and reliable but also exhibits a higher sensitivity.