A primer pair, crRNA and a method for detecting single base drug-resistant mutation of mycoplasma bovis based on RPA-CRISPR / Cas12a

Abstract

This invention belongs to the field of biotechnology, specifically disclosing a primer pair, crRNA, and an RPA-CRISPR / Cas12a-based method for detecting single-base drug resistance mutations in bovine mycoplasma. This invention designs and synthesizes an RPA primer pair for amplifying the A2058G gene mutation site and a specific crRNA targeting the A2058G gene mutation site. Combined with a fluorescent reporter probe, this enables rapid, simple, and specific visual detection of bovine mycoplasma drug resistance. The detection method provided by this invention requires no complex instruments and equipment, is widely applicable to on-site real-time detection, and has good application prospects, providing new methodological support for the rapid determination of bovine mycoplasma drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to a primer pair, crRNA, and a method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a. Background Technology

[0002] Bovine mycoplasma ( Mycoplasma bovis , M. bovis Mycoplasma belongs to the class Mycoplasmata and is a minimal prokaryotic microorganism without a cell wall that can self-replicate. M. bovis Not only is it a major cause of bovine respiratory disease syndrome (BRD), but it also frequently leads to a variety of other diseases such as pneumonia, mastitis, arthritis, keratitis, and otitis media, causing significant economic losses to the global cattle industry. Current clinical treatment... M. bovis Infections are primarily treated with antibiotics such as macrolides, tetracyclines, fluoroquinolones, and aminoglycosides. However, in recent years, globally... M. bovis Drug resistance is becoming increasingly prominent, especially the resistance rate to macrolide antibiotics, which has become a key factor restricting clinical efficacy.

[0003] Studies have shown that M. bovis The main molecular mechanisms of macrolide resistance are concentrated on the modification of drug target sites, with the A2058G point mutation in the V region of the ribosomal 23S rRNA being the most prevalent form of resistance. This site is a key region for macrolide drug binding; mutations at this site significantly reduce the affinity of the drug for the ribosome, leading to high levels of resistance. Traditional pathogen resistance detection mainly relies on drug susceptibility testing, which requires pathogen isolation and culture, as well as determination of the minimum inhibitory concentration (MIC), typically taking 3 to 7 days, making it difficult to meet the needs of rapid clinical diagnosis. While PCR technology has improved the speed of pathogen detection, it still relies on thermal cycling equipment and specialized laboratories. In recent years, the rise of isothermal amplification technology has provided a new approach to overcoming the bottleneck in pathogen resistance detection. Recombinase polymerase isothermal amplification (RPA) is a novel technology that rapidly completes nucleic acid amplification without the need for thermal cycling equipment, offering advantages such as ease of operation, rapid reaction, and suitability for on-site detection. The CRISPR / Cas system, as an adaptive immune mechanism in archaea and bacteria, has shown great potential in the field of molecular diagnostics in recent years. Among them, the Cas12a protein possesses unique "trans-cleavage" activity and a strict tolerance for mismatches in the matching of its crRNA with the target sequence. Utilizing these two characteristics, a highly specific detection platform for distinguishing single nucleotide polymorphisms (SNPs) can be established.

[0004] Combining the amplification capabilities of RPA with the high specificity of CRISPR / Cas12a has become an important development direction for next-generation molecular diagnostic technologies. However, no studies have been reported on the application of RPA-CRISPR / Cas12a technology to the detection of single-base mutation sites of resistance to macrolide antibiotics in bovine mycoplasma. Faced with the increasingly severe drug resistance situation, establishing rapid and accurate drug resistance detection methods is of significant scientific importance and practical value for guiding rational drug use in clinical practice and curbing the spread of drug resistance. Summary of the Invention

[0005] To address the aforementioned problems, this invention aims to establish a method for detecting single-base mutation sites of resistance to macrolide antibiotics in bovine mycoplasma based on RPA-CRISPR / Cas12a technology. This method will enable accurate differentiation between sensitive and resistant strains, solving the prominent problems of long cycles, complex operations, and equipment dependence in current clinical drug resistance testing. It will provide a new technical tool for the accurate diagnosis and treatment of bovine mycoplasma disease and the monitoring of drug resistance.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a primer pair comprising an upstream primer PAM-F6 as shown in SEQ ID NO.12 and a downstream primer PAM-R as shown in SEQ ID NO.6.

[0007] In a second aspect, the present invention provides a crRNA, the sequence of which is shown in SEQ ID NO.15.

[0008] A third aspect of the present invention provides a kit comprising an RPA primer pair for amplifying a gene mutation site A2058G, a specific crRNA targeting the gene mutation site A2058G, and a fluorescent reporter probe. The gene mutation site A2058G is located in the 23S rRNA of bovine mycoplasma ribosomes. rrnA The 2058th base in the V region of the gene, wherein the 2058th base is either A or G; The RPA primer pair used to amplify the gene mutation site A2058G includes the upstream primer PAM-F6 as shown in SEQ ID NO.12 and the downstream primer PAM-R as shown in SEQ ID NO.6; The sequence of the specific crRNA targeting the gene mutation site A2058G is shown in SEQ ID NO.15.

[0009] In a preferred embodiment, the nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO.16.

[0010] In a preferred embodiment, the fluorescent reporter probe is labeled with the fluorescent group FAM at its 5′ end and with the quencher group BHQ1 at its 3′ end.

[0011] A fourth aspect of the present invention provides a method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a, characterized by comprising the following steps: S1. Extract genomic DNA from bovine mycoplasma samples; S2. Using the genomic DNA extracted in step S1 as a template, RPA amplification is performed using the upstream primer PAM-F6 shown in SEQ ID NO.12 and the downstream primer PAM-R shown in SEQ ID NO.6 to obtain the RPA amplification product. S3. The RPA amplification product obtained in step S2 is detected using a CRISPR / Cas12a system including crRNA6 as shown in SEQ ID NO.15 and ssDNAreporter as shown in SEQ ID NO.16. The detection results are obtained using a fluorescence signal detector or chromatography strip.

[0012] Under the preferred scheme, the RPA amplification reaction conditions are: constant temperature reaction at 39℃ for 20 min.

[0013] In a preferred embodiment, the reaction system of the CRISPR / Cas12a system comprises: 2 μL of 10×Reaction Buffer, 1 μL of Cas12a protein, 0.6 μL of ssDNA reporter, 1 μL of RPA amplification product, and DEPC water to a final volume of 20 μL.

[0014] In the preferred embodiment, when obtaining the detection results using a fluorescence signal detector, the concentration of ssDNA reporter in the CRISPR / Cas12a system reaction system is 200 nM, the concentration of Cas12a protein is 110 nM, and the concentration of crRNA6 is 50 nM; the reaction conditions for step S3 are: constant temperature reaction at 37℃ for 1 h.

[0015] In the preferred embodiment, when obtaining the test results using the chromatographic test strip, the concentration of ssDNA reporter is 60 nM; the reaction conditions for step S3 are: constant temperature reaction at 37℃ for 40 min.

[0016] Beneficial effects of the present invention This invention designs and synthesizes RPA primer pairs for amplifying the A2058G gene mutation site. By combining RPA amplification technology, a CRISPR / Cas12a specific recognition system, and a double-antibody sandwich lateral flow strip (LFS) detection technology, a rapid molecular diagnostic platform suitable for the A2058G macrolide resistance mutation site in Mycoplasma bovis is constructed. This invention also designs and synthesizes crRNA targeting the A2058G gene mutation site, which can accurately distinguish between drug-resistant and drug-sensitive Mycoplasma bovis sequences. The RPA-CRISPR / Cas12a two-step detection system is optimized, stable reaction conditions are established, and nucleic acid strip technology is used to achieve visualized interpretation of the results.

[0017] This invention combines RPA technology with a CRISPR / Cas12a detection system, achieving integrated detection of isothermal amplification, specific recognition, and signal output. It boasts advantages such as high sensitivity, strong specificity, and ease of operation. Furthermore, a rapid detection technique can be developed that does not rely on complex instruments such as agarose gel electrophoresis, fluorescence imaging systems, or real-time quantitative PCR instruments, providing new methodological support for the rapid determination of bovine mycoplasma drug resistance. Attached Figure Description

[0018] Figure 1 This invention describes the detection principle of the RPA-CRISPR / Cas12a system.

[0019] Figure 2 This is a schematic diagram of the RPA primer pair design of the present invention.

[0020] Figure 3 The RPA amplification results of the RPA primer pair PAM-F / PAM-R of the present invention are shown; wherein, M: DL2000 Marker, 1: genomic DNA of bovine mycoplasma standard strain PG45, 2: pMB-R, 3: pMB-S, 4: negative control.

[0021] Figure 4 The RPA amplification results of the RPA primer pairs of this invention are shown below. Templates 1, 3, 5, 7, 9, and 11 are pMB-R; 2, 4, 6, 8, 10, and 12 are pMB-S; N is the negative control. RPA primer pairs are: 1-2: PAM-F1 / PAM-R; 3-4: PAM-F2 / PAM-R; 5-6: PAM-F3 / PAM-R; 7-8: PAM-F4 / PAM-R; 9-10: PAM-F5 / PAM-R; 11-12: PAM-F6 / PAM-R.

[0022] Figure 5 The sequencing analysis results of the six RPA amplification products of this invention are shown.

[0023] Figure 6 This is the optimization result of the reaction temperature of the PAM-F1 / PAM-R primer pair of the present invention; wherein, templates 1, 3, 5, 7, and 9 are pMB-R, 2, 4, 6, 8, and 10 are pMB-S, and N is the negative control; reaction temperature: 1-2: 25℃, 3-4: 29℃, 5-6: 34℃, 7-8: 39℃, 9-10: 44℃.

[0024] Figure 7 The results show the optimized reaction time of the RPA primer pair PAM-F1 / PAM-R in this invention; wherein, templates 1, 3, 5, and 7 are pMB-R, 2, 4, 6, and 8 are pMB-S, and N is the negative control; reaction temperature: 1-2: 10 min, 3-4: 15 min, 5-6: 20 min, 7-8: 25 min.

[0025] Figure 8 The feasibility analysis results of crRNA1 designed for this invention are as follows: A: qPCR fluorescence curve results; B: endpoint fluorescence reaction tube results; C: endpoint fluorescence value analysis for each group.

[0026] Figure 9 The feasibility analysis results for the crRNA4 designed for this invention are as follows: A: qPCR fluorescence curve results; B: endpoint fluorescence reaction tube results; C: endpoint fluorescence value analysis for each group.

[0027] Figure 10 The feasibility analysis results of the crRNA6 designed in this invention; wherein, A: qPCR fluorescence curve results; B: endpoint fluorescence reaction tube results; C: endpoint fluorescence value analysis of each group.

[0028] Figure 11 The results of the false positive analysis of the crRNA6 reaction designed in this invention are shown below; where A: fluorescence curve results output by qPCR instrument; B: results of endpoint fluorescence reaction tube; C: analysis of endpoint fluorescence values ​​for each group.

[0029] Figure 12 The results of the optimized analysis of the concentration of the fluorescent reporter probe of this invention are shown below; where A: fluorescence curve results output by the qPCR instrument; B: results of the endpoint fluorescence reaction tube; C: analysis of the endpoint fluorescence values ​​of each group.

[0030] Figure 13 The results of the optimized analysis of Cas12a protein concentration in this invention are shown below; where A: fluorescence curve results output by qPCR instrument; B: results of endpoint fluorescence reaction tubes; C: analysis of endpoint fluorescence values ​​for each group.

[0031] Figure 14The results of the optimized analysis of crRNA6 concentration in this invention are shown below; where A: fluorescence curve results output by qPCR instrument; B: results of endpoint fluorescence reaction tubes; C: analysis of endpoint fluorescence values ​​for each group.

[0032] Figure 15 The results of the optimization analysis of the test strip detection method of the present invention are as follows: A: optimization results of R-DNA test strip type report probe; B: optimization results of S-DNA test strip type report probe; C: optimization results of test strip detection time.

[0033] Figure 16 The results of sensitivity detection analysis of the RPA-CRISPR / Cas12a system of the present invention are as follows: A: fluorescence curve results of qPCR instrument; B: results of endpoint fluorescence reaction tube; C: analysis of endpoint fluorescence values ​​of each group; D: test strip test results.

[0034] Figure 17 The results of the specific detection and analysis of the RPA-CRISPR / Cas12a system of this invention are as follows: A: qPCR fluorescence curve results; B: endpoint fluorescence signal results; C: analysis of endpoint fluorescence values ​​for each group; D: test strip results (1-7: drug-resistant Mycoplasma bovis Mb1; Proteus mirabilis; Hemolytic Mansonia; Pasteurella multocida; Salmonella; Staphylococcus aureus; Klebsiella pneumoniae; negative control).

[0035] Figure 18 The results of fluorescence detection analysis of bovine mycoplasma samples are shown below; where A and B are the fluorescence curve results of qPCR instrument; C is the endpoint fluorescence signal result; and D is the analysis of the endpoint fluorescence value of each group.

[0036] Figure 19 The results of the test strip analysis for bovine mycoplasma samples.

[0037] In the figure, different letters indicate that there are significant differences in the levels between the groups, p < 0.05. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0039] Example 1: Identification of bovine mycoplasma strain phenotype and drug resistance gene mutation sites 1. Phenotypic identification of bovine mycoplasma strains Pathological samples were collected from cattle suffering from bovine respiratory disease syndrome (BRS). After isolation and identification, 32 Mycoplasma bovis strains were obtained. Antibiotic susceptibility testing was conducted using the microdilution method to determine the susceptibility of the 32 isolated Mycoplasma bovis strains to macrolide antibiotics (erythromycin, azithromycin, and tylosin). The results showed that 13 Mycoplasma bovis strains were resistant (MIC ≥ 64 μg / mL), and 19 Mycoplasma bovis strains were susceptible (MIC = 32 μg / mL or MIC ≤ 16 μg / mL).

[0040] 2. Identification of drug resistance gene mutation sites in bovine mycoplasma strains 2.1 PCR primer pair design The gene mutation site targeted by this invention is located in the 23S rRNA of the ribosome. rrnA The mutation site at nucleotide position 2058 in region V of the gene is an A→G mutation site, named A2058G. The bovine mycoplasma 23S rRNA sequence NC_014760.1[322911.325800] was downloaded from the NCBI database and analyzed using SnapGene software. The bovine mycoplasma 23S rRNA was found. rrnA Specific primers MB23s-F (5′-GTGATGTATAGGGGGTGAAGCC-3′, SEQ ID NO.1) and MB23s-R (5′-CTGACCGTACTGAGTGTACCTTTG-3′, SEQ ID NO.2) were designed for the conserved region of the A2058G mutation site of the gene. The specificity of the designed primers was verified using primer-BLAST from NCBI. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] 2.2 DNA extraction from bovine mycoplasma strains Genomic DNA was extracted from 32 bacterial strains using a boiling method. The specific steps included: taking 1 mL of bacterial culture, centrifuging at 6000 rpm for 2 min, discarding the supernatant, adding 1 mL of ddH2O to resuspend the bacterial pellet, centrifuging at 6000 rpm for 2 min, and discarding the supernatant. Then, adding 100 μL of ddH2O to the bacterial pellet, washing and resuspending the bacterial cells, and after the cells dissolved, heating in boiling water for 10 min, vortexing for 30 s. Centrifuging at 10,000 rpm for 5 min, and collecting the supernatant to obtain the crude DNA extract.

[0042] 2.3 PCR amplification and product sequencing Using DNA extracted from 32 bacterial strains as templates, a gene fragment of approximately 500 bp, including the mutation site upstream and downstream, was amplified by PCR. The PCR reaction system consisted of: 50 μL of 2×Taq PCR Master Mix, 5 μL (10 µM) of MB23s-F, 5 μL (10 µM) of MB23s-R, 3 μL of Template, and ddH2O to a final volume of 100 μL. The PCR reaction conditions were: 95℃ for 3 min; 95℃ for 30 s; 55℃ for 30 s; 72℃ for 20 s; 72℃ for 5 min, for 30 cycles.

[0043] After the amplification reaction was completed, the amplification products from 32 tubes were subjected to 1% agarose gel electrophoresis to confirm that the size of each amplified band was as expected and that the bands were single and free of impurities. A suitable amount of sample from each of the 32 tubes of amplification products, along with primer MB23s-F, was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were used to verify the specific base types of the drug resistance gene mutation sites in each strain.

[0044] Sequencing verification and sequence alignment analysis were performed on the amplified products containing the target fragment. Thirteen drug-resistant strains were found to have 23S rRNA... rrnA The 2058th base position in gene V region was guanine G, while in 19 susceptible strains it was adenine A. The drug resistance phenotypes of 32 bovine Mycoplasma strains were correlated with 23S rRNA. rrnA The point mutation at position 2058 of the gene, A→G, corresponds completely. This mutation site is stable and can serve as reliable information for distinguishing between drug-resistant and drug-sensitive strains.

[0045] Example 2: Construction of standard plasmids for drug-resistant and susceptible bovine mycoplasma strains Based on the sequencing results of Example 1, PCR amplification products from one drug-resistant strain and one susceptible strain were selected. The remaining PCR products were purified and recovered using a product purification kit to obtain purified DNA fragments. M.bovis R Gene fragments (drug-resistant strains) and M.bovis S Gene fragment (sensitive strain). Concentration and purity were determined using NanoDrop (A260 / A280≈1.8, A260 / A230>2.0). 100 ng M.bovis R Gene fragments and M.bovis S The gene fragments were ligated with the pMD18-T vector. The ligation reaction system (10 μL) included: 1 μL pMD18-T vector, 1 μL Control Insert, 5 μL Solution I, and 3 μL ddH2O. The ligation reaction conditions were: 16℃, 30 min.

[0046] After the ligation reaction was completed, the entire reaction mixture was added to 100 μL of DH5α competent cells and plated onto LB agar plates containing Amp resistance. The plates were incubated overnight at 37°C. After single colonies grew on the plates, single colonies were picked and streaked onto new LB agar plates containing Amp resistance using the zigzag method, and incubated at 37°C for 7–8 h. Once single colony culture was complete, colony PCR was performed using universal primers M13-F (5′-TGTAAAACGACGGCCAGT-3′, SEQ ID NO.3) and M13-R (5′-CAGGAAACAGCTATGACC-3′, SEQ ID NO.4) for verification. Agarose gel electrophoresis was used to analyze band size and screen for candidate transformants with the correct band size. Plasmids were extracted from the candidate strains and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Sequencing results showed that the constructed sensitive recombinant plasmid pMB-S was successfully isolated in 23S rRNA. rrnA The base at gene 2058 is A, and the constructed drug-resistant recombinant plasmid pMB-R has a base at the same site, which is G. The base type is completely consistent with the experimental design. The above two plasmids will be used as positive criteria for the subsequent establishment and performance evaluation of the RPA-CRISPR / Cas12a detection system.

[0047] Example 3: Design, Validation and Screening of RPA Primer Pairs 1. Design of RPA primer pairs In the CRISPR / Cas12a system for detecting single-base mutation sites, the selection of the PAM site is crucial for determining detection specificity and differentiating between sensitive and resistant strains. Since there is no suitable PAM site near the A2058G gene mutation site, the 5'-TTT-3' sequence needs to be introduced into the amplification product using an RPA reaction. Before introducing this sequence, the position of the RPA primers should be carefully selected to ensure that the primer specificity and amplified fragment length meet the requirements of subsequent experiments. Following the RPA primer design principles, the sequence of Bovine Mycoplasma 23S rRNA NC_014760.1[322911.325800] was analyzed using SnapGene software. The Bovine Mycoplasma 23S rRNA was found... rrnA Primers PAM-F and PAM-R were designed at the A2058G mutation site of the gene, located in its conserved region, as shown in the figure. Figure 2 As shown in Table 1, the specificity of the designed primers was verified using primer-BLAST from NCBI. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] Table 1: RPA primer pair sequences

[0049] 2. Validation of RPA primer pairs To detect the amplification effect of the RPA primers, the RPA isothermal amplification kit (Anpu Future Biotechnology Co., Ltd.) and the PAM-F / PAM-R primer pair were used to perform RPA amplification reactions on the genomic DNA of the bovine mycoplasma standard strain PG45 (provided and preserved by the Veterinary Pharmacology Laboratory of Jilin Agricultural University), the recombinant plasmids pMB-R and pMB-S constructed in Example 2.

[0050] First, add 29.4 μL of buffer A to each dry powder reaction tube and mix thoroughly. Then, add 2 μL each of the upstream and downstream primers PAM-F / PAM-R, with a final primer concentration of 10 μM. Add 5 μL of genomic DNA as template and 9.1 μL of ddH2O to each reaction system, and include a negative control without plasmid template. Next, add 2.5 μL of buffer B to each reaction tube and mix thoroughly. After mixing, briefly centrifuge to collect the liquid at the bottom of the tube, and incubate at 37°C for 30 min as soon as possible. After the reaction, add 50 μL of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) DNA extraction buffer to the product, mix thoroughly, and centrifuge at 12000 rpm for 5 min. Finally, take 5 μL of the supernatant, mix with 1 μL of 6× Loading Buffer, and perform 2% agarose gel electrophoresis. The results are shown below. Figure 3 As shown.

[0051] Experimental results showed that using PAM-F / PAM-R as the RPA primer pair, RPA amplification of the genomic DNA of the bovine mycoplasma standard strain PG45, recombinant plasmids pMB-R and pMB-S, all produced bright and single bands. The negative control showed no amplified band, indicating that the RPA primer pair PAM-F / PAM-R has good specificity and amplification efficiency, and can be used for the design of subsequent PAM site introduction.

[0052] 3. Insertion of PAM sites The Cas12a protein recognizes the PAM site: 5'-TTTV-3' (where V represents A, C, or G). Based on experimental verification that primers in this region possess good specificity and amplification efficiency, and considering the difference in the number of bases between the PAM site and the target drug-resistant mutation site, the PAM recognition site 5'-TTT-3' was directionally inserted into the corresponding position of the forward primer PAM-F to meet the Cas12a protein's requirements for recognizing and cleaving the target DNA. Following RPA primer design principles, six RPA primers were designed using SnapGene software. The complete primer sequences are shown in Table 2. The underlined bolded region, representing the PAM site insertion position, was synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.

[0053] Table 2: RPA upstream primer sequences after PAM insertion site

[0054] 4. Screening of RPA primer pairs and sequencing of amplification products 4.1 Screening of RPA primer pairs The six upstream RPA primers designed in Table 2 for insertion into the PAM site were combined with their respective downstream primers PAM-R to obtain six RPA primer pair combinations (PAM-F1 / PAM-R, PAM-F2 / PAM-R, PAM-F3 / PAM-R, PAM-F4 / PAM-R, PAM-F5 / PAM-R, and PAM-F6 / PAM-R). The six RPA primer pairs were amplified using the same RPA amplification system (RPA system and reaction conditions as verified above), and target DNA fragments of approximately 200 bp were amplified using the constructed drug-resistant recombinant plasmid pMB-R and the sensitive recombinant plasmid pMB-S as templates. After amplification, the products of each group were detected by 2% agarose gel electrophoresis, and the amplification effect of each primer pair was comprehensively evaluated based on the presence, size, and specificity of the electrophoretic bands. The screening criteria included the ability to amplify a single, non-specific band, a fragment size consistent with the expected 200 bp, and clear band brightness. Preliminary screening identified qualified RPA candidate primer pairs with good amplification performance. The screening results are as follows: Figure 4 As shown.

[0055] The results showed that among the six RPA primer pairs, PAM-F1 / PAM-R, PAM-F4 / PAM-R, and PAM-F6 / PAM-R all amplified a single, clear target band of the expected size, with little or no non-specific amplification. The other three primer pairs (PAM-F2 / PAM-R, PAM-F3 / PAM-R, and PAM-F5 / PAM-R) had lower amplification efficiency or produced non-specific bands. Based on the screening results, PAM-F1 / PAM-R, PAM-F4 / PAM-R, and PAM-F5 / PAM-R were initially selected as candidate primer pairs for subsequent reaction condition optimization and experiments on the CRISPR / Cas12a detection system.

[0056] 4.2 Sequencing of amplified products The RPA amplification products obtained by the above-mentioned candidate primer pairs (including the R-DNA amplification product using the drug-resistant recombinant plasmid pMB-R as a template and the S-DNA amplification product using the sensitive recombinant plasmid pMB-S) were sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. After sequencing, the sequencing sequence was compared with the target mutant sequence using SnapGene software, focusing on whether the PAM site was accurately introduced to verify the correctness of the target sequence and whether the positions of the mutant site and the PAM site met the requirements of the subsequent Cas12a system.

[0057] Table 3: Information on candidate primer pairs and amplification products

[0058] Sequencing results as follows Figure 5 As shown, the sequences of all six amplification products are consistent with the expected design, and the PAM site (5'-TTTV-3') has been successfully introduced into the corresponding positions of the S-DNA and R-DNA sequences.

[0059] Example 4: Optimization of RPA amplification reaction conditions To further improve the amplification efficiency of the amplification system and reduce the generation of nonspecific amplification products, optimization experiments were conducted on two key influencing parameters in the RPA reaction process: reaction temperature and reaction time.

[0060] 1. Optimization of reaction time Using PAM-F1 / PAM-R, and with recombinant plasmids pMB-R and pMB-S as templates, five temperature gradients (25℃, 29℃, 34℃, 39℃, and 44℃) were established. The remaining procedures and conditions followed the steps in Example 3, "Verification of RPA Primer Pairs," for isothermal RPA amplification. After the reaction, 5 μL of the amplification product was subjected to 2% agarose gel electrophoresis. Based on the brightness of the target band and the non-specific amplification in the electrophoresis results, the amplification results under each temperature condition were analyzed. The temperature with the highest amplification efficiency and no non-specific bands was selected as the optimal reaction temperature. The results are shown below. Figure 6 As shown.

[0061] like Figure 6 As shown, among the five temperature gradients, the target band obtained at 39℃ was the clearest and brightest, and no obvious non-specific amplification was observed. Therefore, 39℃ was determined as the optimal reaction temperature.

[0062] 2. Optimization of reaction time Using PAM-F1 / PAM-R, and recombinant plasmids pMB-R and pMB-S as templates, RPA isothermal amplification was performed at the determined optimal reaction temperature of 39℃, with four time gradients of 10 min, 15 min, 20 min, and 25 min. The remaining procedures and conditions followed the steps in Example 3, "Verification of RPA Primer Pairs." After the reaction, the amplification products were detected by 2% agarose gel electrophoresis, observing the appearance and brightness changes of the target band at each time point. The shortest reaction time that stably amplified a clear target band was taken as the optimal reaction time. The results are as follows: Figure 7 As shown.

[0063] like Figure 7 As shown, comparisons under different reaction time conditions revealed that a clear target band could be stably amplified after 20 minutes of reaction. Therefore, 20 minutes was selected as the optimal reaction time. Based on the above results, this study ultimately determined the optimal conditions for RPA amplification to be a constant temperature reaction at 39℃ for 20 minutes.

[0064] Example 5: Design, screening, and false positive verification of crRNA 1. Design of crRNA Based on the R-DNA and S-DNA sequences of the amplified products after successful insertion of the PAM site, as confirmed by sequencing ( Figure 5To target the key macrolide resistance mutation site A2058G in the bovine mycoplasma 23S rRNA gene, a crRNA for CRISPR / Cas12a detection was designed. Based on crRNA design principles, three candidate crRNAs were designed: crRNA1, crRNA4, and crRNA6. The crRNA with the best recognition efficiency and specificity was selected in subsequent experiments. The sequences of the three crRNAs with mismatched bases are shown in Table 4. In the table, the bolded region represents the hairpin structure of the crRNA, and "U" indicates an introduced mismatched base.

[0065] Table 4: Design of crRNA sequences

[0066] 2. Establishment of the RPA-CRISPR / Cas12a system and screening of crRNA The ssDNA fluorescent reporter probe used in the system has the sequence 5'-TTTAATTT-3' (SEQ ID NO. 16), with the fluorescent group being FAM and the quenching group being BHQ1. It was synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.

[0067] The Cas12a system buffer (10×Reaction Buffer) was prepared as follows: 500mM NaCl, 100mM Tris-HCl, 100mM MgCl2·6H2O, 1mg / mL BSA, pH=7.9.

[0068] Using four key components—amplification products, Cas12a protein, crRNA, and ssDNA fluorescent reporter probe—as variables, three control experiments were designed to detect the CRISPR / Cas12a reactivity of different target combinations: R-DNA1 / S-DNA1 amplification products with crRNA1; R-DNA4 / S-DNA4 amplification products with crRNA4; and R-DNA6 / S-DNA6 amplification products with crRNA6. The CRISPR / Cas12a reaction system (20 μL) is shown in Table 5.

[0069] Table 5: CRISPR / Cas12a reaction system

[0070] The reaction mixture was added to an eight-tube strip, briefly centrifuged to ensure thorough mixing at the bottom, and then placed in a quantitative PCR instrument to detect the fluorescence signal. All steps were performed on ice. The instrument program was set as follows: the FAM channel was selected as the reporter fluorescence signal, quenching signal acquisition was turned off, the hot cap function was turned off, the reaction temperature was 37°C, the signal acquisition interval was set to once every 60 seconds, for a total of 60 cycles, and the total reaction time was 1 hour. After the reaction, the fluorescence signal value change curve during the reaction was analyzed using a quantitative PCR instrument to confirm the cleavage activity of the CRISPR / Cas12a system. Simultaneously, the reaction tube was placed in a multifunction developing instrument to determine the presence and intensity of the endpoint fluorescence. The overall activity of the detection system was verified by combining the detection results, and the optimal-performing crRNA was screened.

[0071] The validation results of the amplified products R-DNA1 / S-DNA1 and crRNA1 are as follows: Figure 8 As shown, the fluorescence curve of the R-DNA1 group showed a continuous upward trend from the start of the reaction, with a strong fluorescence signal in the reaction tube at the endpoint; the fluorescence curve of the S-DNA1 group only began to rise slowly after 20 minutes of reaction, and the reaction tube showed weak fluorescence; the control group, which lacked key reaction components, did not show any fluorescence signal. This result indicates that the constructed CRISPR / Cas12a system is functionally complete and normally activated. However, since both R-DNA1 and S-DNA1 showed different degrees of fluorescence reaction in this system, it indicates that crRNA1 still cannot accurately distinguish between drug-resistant and sensitive sequences. Therefore, crRNA1 was not selected as the recognition RNA for detecting macrolide resistance mutation sites in bovine mycoplasma in this study.

[0072] The validation results of the amplified products R-DNA4 / S-DNA4 and crRNA4 are as follows: Figure 9 As shown, the fluorescence curves of the R-DNA4 and S-DNA4 groups continuously increased from the start of the reaction, and the reaction tubes corresponding to the reaction endpoint all showed strong fluorescence signals, while the control group lacking other key components did not show obvious fluorescence reactions. However, both R-DNA4 and S-DNA4 in this group showed significant fluorescence signals, and the endpoint fluorescence values ​​were both large. crRNA4 could not effectively distinguish between R-DNA and S-DNA sequences. Therefore, crRNA4 was not used as the recognition RNA for detecting macrolide resistance mutation sites in bovine mycoplasma in this study's CRISPR / Cas12a system.

[0073] The validation results of the amplified products R-DNA6 / S-DNA6 and crRNA6 are as follows: Figure 10As shown, the fluorescence intensity of the R-DNA6 group increased continuously from the start of the reaction, and the reaction tubes corresponding to the reaction endpoint all showed strong fluorescence signals. In contrast, the S-DNA6 group and all control groups lacking key reaction components did not show significant fluorescence reactions or fluorescence curve increases. The endpoint fluorescence value of the R-DNA6 group was significantly higher than that of the other groups, indicating that crRNA6 can specifically recognize the target drug-resistant sequence R-DNA6 and effectively activate the trans-cleavage activity of the Cas12a protein. It can also completely distinguish between drug-resistant and sensitive sequences. Therefore, crRNA6 was selected as the target crRNA for specifically recognizing macrolide resistance mutation sites in bovine mycoplasma in this study's CRISPR / Cas12a detection system.

[0074] In summary, the results of the three sets of control experiments showed that the drug-resistant amplification products R-DNA in all three groups could produce strong fluorescent signals, while the DNA-, Report-, crRNA-, and Cas12a- groups could not produce fluorescent signals. This indicates that the purified Cas12a protein and the in vitro transcribed crRNA in this study both have normal biological activity, and a stable and reliable CRISPR / Cas12a detection system was successfully constructed. Among them, crRNA1 and crRNA4 could not effectively distinguish between drug-resistant and sensitive sequences due to the non-specific fluorescence of S-DNA, while crRNA6 could specifically activate R-DNA and completely inhibit the S-DNA fluorescent signal, making it the best crRNA. Its matching PAM-F6 / PAM-R is the optimal primer pair.

[0075] 2. Verification of crRNA positivity Based on the CRISPR / Cas12a system validation results, and in conjunction with different concentrations of amplified DNA template, the study analyzed whether the concentration of the sensitive plasmid pMB-S affected the CRISPR / Cas12a reaction activity of R-DNA6 / S-DNA6 and crRNA6. In the RPA amplification step, three concentration gradients of the amplified template pMB-S were set: 500 ng / μL, 100 ng / μL, and 10 ng / μL; the concentration of the amplified template pMB-R was 10 ng / μL. A negative control (without amplified template) was also included. RPA isothermal amplification was performed using primer pairs PAM-F6 / PAM-R with the above template (the process and conditions were the same as in Example 3, "Validation of RPA Primer Pairs"). The RPA amplification product was then reacted with crRNA6 using CRISPR / Cas12a (the process and conditions were the same as described in this example). Changes in the fluorescence curve during the reaction were detected using a quantitative real-time PCR instrument, and the endpoint fluorescence of the reaction tubes was observed.

[0076] False positive verification results of the CRISPR / Cas12a reaction of R-DNA6 / S-DNA6 with crRNA6 are as follows: Figure 11As shown, the S-DNA6 fluorescence curves amplified using pMB-S at concentrations of 500 ng / μL, 100 ng / μL, and 10 ng / μL as templates did not rise, and no fluorescence development was observed in the reaction tubes; the R-DNA6 fluorescence curve amplified using pMB-R at a concentration of 10 ng / μL as template showed a significant rise from the start of the reaction, with clear fluorescence development in the reaction tubes and a high fluorescence value at the endpoint; no obvious fluorescence signal was detected in the negative control group.

[0077] Example 6: Optimization of the RPA-CRISPR / Cas12a System Based on the experimental results of Example 5 above, the primer pair with the highest amplification efficiency and specificity was selected and combined with crRNA. This combination can perform efficient RPA isothermal amplification of pMB-R and pMB-S plasmids, and the corresponding CRISPR / Cas12a system can specifically recognize and distinguish between S-DNA and R-DNA amplification products. Based on the determined optimal primer pair and crRNA combination, the key parameters of the CRISPR / Cas12a system were systematically optimized, including the concentration of the fluorescent reporter probe, the working concentration of Cas12a protein, and the working concentration of crRNA. All amplification products added to the detection system were obtained by RPA amplification using 1 ng / μL of pMB-R as a template.

[0078] 1) Optimization of fluorescent reporter probe concentration: Set up four probe concentration gradients of 50nM, 100nM, 150nM and 200nM, keep the other reaction components constant, and set up a blank control without probe.

[0079] 2) Optimization of Cas12a protein working concentration: Four protein concentration gradients were set at 20 nM, 50 nM, 80 nM, and 110 nM, while the other reaction components were kept constant. A blank control without protein was also set up.

[0080] 3) Optimization of crRNA working concentration: Set up three crRNA concentration gradients of 20nM, 50nM and 80nM, keep the other reaction components constant, and set up a blank control without crRNA.

[0081] The optimization results of the fluorescent reporter probe concentration are as follows: Figure 12 As shown in the figure. This experiment used the real-time fluorescence curve of the qPCR instrument and the endpoint fluorescence color development of the reaction tube as the analytical standards to compare the effects of different concentrations of fluorescent reporter probes on detection performance. The results showed that the overall fluorescence signal intensity in the system increased with increasing probe concentration. Among them, the 200 nM group not only had the most significant endpoint fluorescence signal, but also a faster rate of fluorescence curve rise. Therefore, 200 nM was determined to be the optimal fluorescent reporter probe concentration for this CRISPR / Cas12a detection system.

[0082] The optimization results of Cas12a protein concentration are as follows: Figure 13 As shown, this study systematically evaluated the effect of different concentrations of Cas12a protein on the performance of the CRISPR / Cas12a detection system. The results showed that the fluorescence signal intensity gradually increased with increasing Cas12a protein concentration, with the 110 nM Cas12a protein concentration group exhibiting the best performance. Its endpoint fluorescence signal value was significantly higher than other concentration groups, and the fluorescence curve rose at a faster rate. The other concentration groups all showed weak fluorescence responses. Considering both detection sensitivity and reaction efficiency, 110 nM was ultimately determined to be the optimal Cas12a protein concentration for this CRISPR / Cas12a detection system.

[0083] The optimization results of crRNA concentration are as follows: Figure 14 As shown, the performance of different concentrations of crRNA was evaluated using real-time fluorescence curves recorded by a qPCR instrument and the final fluorescence signal of the reaction tubes. The fluorescence signal intensities of each group were very similar; the fluorescence signal value of 20 nM crRNA was almost the same as that of 80 nM. The fluorescence curve of the 50 nM crRNA group increased rapidly from the start of the reaction, and the final fluorescence signal value was the strongest. Strong fluorescence reactions were produced in all three reaction tubes, ultimately determining 50 nM as the optimal crRNA concentration for this CRISPR / Cas12a detection system.

[0084] Based on the comprehensive analysis of the above experimental results, the optimal reaction conditions for the RPA-CRISPR / Cas12a fluorescence detection system were determined as follows: fluorescent probe concentration of 200 nM, Cas12a protein concentration of 110 nM, and crRNA concentration of 50 nM. Under these optimal conditions, the detection system exhibits the best detection capability, thus laying a solid foundation for the rapid detection of mutation sites.

[0085] Example 7: Establishment and optimization of CRISPR / Cas12a test strip detection method 1. Establishment of CRISPR / Cas12a test strip detection method The CRISPR / Cas12a test strip detection method used in this experiment is based on chromatographic double-antibody sandwich immunochromatography technology, enabling rapid and visual detection of reporter molecules with dual labeling modifications. The test strip-type reporter probe sequence used is 5'-TTTAATTT-3', with FAM fluorescent group and biotin group labeled at both ends, respectively, which can be specifically degraded by Cas12a trans-cleavage.

[0086] When R-DNA is present in the detection system, the ribonucleoprotein complex formed by the binding of Cas12a protein and crRNA can be specifically activated, thereby initiating trans-cleavage activity and fully degrading the dual-labeled reporter probe in the system. After the reporter molecule is cleaved, it cannot form an immune sandwich complex on the test line (T line), so no band appears on the T line, while the control line (C line) shows normal color, and the result is judged as positive; if only a weak band appears on the T line and the C line shows normal color, it is judged as weakly negative. Conversely, when only S-DNA is present in the system or there is no target template, the Cas12a protein cannot be activated, the reporter molecule retains its intact structure, and can form specific bands on both the test line and the control line simultaneously, that is, both the T line and the C line show normal color, and the result is judged as negative.

[0087] Based on the above principles, this study systematically proportioned the concentrations and reaction volumes of each component in the CRISPR / Cas12a test strip detection system, establishing a stable and visualized detection reaction system. The specific composition of the reaction system is shown in Table 6.

[0088] Table 6. CRISPR / Cas12a test strip detection system (20 μL)

[0089] Add 20 μL of the reaction mixture to an eight-tube strip, briefly centrifuge to ensure the solution is thoroughly mixed at the bottom of the tube, and incubate at 37°C in a metal bath for 1 hour. Take 2 μL of the reaction product and add it to 78 μL of Diluent Buffer, mix well. Add 70 μL of the diluted reaction product to the sample well of the test strip, read and record the colorimetric results in the detection area within 5 minutes, and take a photograph.

[0090] 2. Optimization of CRISPR / Cas12a test strip detection method When using this test strip detection method, the amount of reporting probe needs to be optimized. Excessive probe concentration may lead to insufficient probe cleavage, resulting in false negative results; insufficient concentration may result in insufficient cleavage signal, leading to false positive results. Simultaneously, the reaction time needs to be systematically optimized to minimize the detection time while ensuring the reporting probe is fully cleaved by Cas12a, thus achieving rapid and accurate interpretation.

[0091] Optimization of probe concentration for test strip reports: Six probe concentration gradients of 20nM, 40nM, 60nM, 80nM, 100nM, and 120nM were set up, while other reaction components remained constant. A blank control without probes was also included. All amplification products added to the detection system were obtained by RPA amplification using 1 ng / μL of pMB-R and pMB-S plasmids as templates.

[0092] Optimization of test strip reaction time: Five reaction time gradients were set at 10 min, 20 min, 30 min, 40 min, and 50 min, while other reaction components remained constant. All amplification products added to the detection system were obtained by RPA amplification using 1 ng / μL of pMB-R plasmid as a template.

[0093] The optimization results of probe concentration for test strip reports are as follows: Figure 15 As shown, Figure 15 A represents the test strip result of the reaction between R-DNA6 and crRNA6. Figure 15 B represents the test results of the S-DNA6 and crRNA6 reaction strips. When the concentration of the report probe is higher than 80 nM, the T line of the R-DNA group test strip cannot be completely eliminated, resulting in a negative result, while the T line of the S-DNA group test strip develops normally. This result cannot distinguish between R-DNA and S-DNA. When the concentration of the report probe is 60 nM, the T line of the R-DNA group test strip disappears completely, while the T line of the S-DNA group test strip develops normally. When the concentration of the report probe is 40 nM, the S-DNA group test strip shows a fading color, which can easily lead to false positives. Therefore, considering both reaction results, 60 nM is selected as the optimal report probe concentration for the test strip detection.

[0094] The optimization results of the reaction time for the test strip are as follows: Figure 15 As shown in Figure C, the T-line of the test strip develops color when the reaction time is 10 min and 20 min; the color of the T-line weakens when the reaction time is 30 min; and the T-line disappears completely when the reaction time is 40 min and 50 min. Therefore, 40 min is selected as the optimal reaction time for the test strip detection.

[0095] The results in summary indicate that the optimal conditions for the test strip detection method are: a 60 nM test strip reporter probe concentration and a 40-minute reaction time. These conditions ensure that the reporter probe is fully cleaved by the Cas12a protein while shortening the detection time, thus achieving rapid and accurate interpretation.

[0096] Example 8: Sensitivity Test of RPA-CRISPR / Cas12a System 1. Test Methods To verify the sensitivity of the two established RPA-CRISPR / Cas12a detection methods for detecting the macrolide resistance mutation site (A2058G) in bovine mycoplasma, a sensitivity test was performed using the resistant pMB-R plasmid as a template. The quantified mutant plasmid template was serially diluted 10-fold to obtain the following concentration gradients: 1.8 × 10⁻⁶ 8 1.8×10 7 1.8×10 6 1.8×105 1.8×10 4 1.8×10 3 1.8×10 2 At concentrations of 1.8 × 10⁸ copies / μL, under the optimal reaction conditions determined in the previous optimization, RPA-CRISPR / Cas12a was sequentially applied to samples of each concentration gradient. The results were interpreted as follows: 1) Fluorescence curve: The reaction system was placed in a real-time quantitative PCR instrument, and the fluorescence signal was recorded every 60 seconds during the reaction at 37℃ for 1 hour. After the reaction, the fluorescence signal change curves of different template concentrations were observed.

[0097] 2) Fluorescence reaction: After the reaction is completed, the reaction tubes of each concentration gradient sample are placed under a multi-functional developing instrument to observe and photograph the endpoint fluorescence reaction, and the difference in fluorescence signal intensity corresponding to different template concentrations is interpreted.

[0098] 3) Strip colorimetric interpretation: Mix the product from the CRISPR-Cas12a reaction with the test strip's detection buffer, add the mixture to the test strip for chromatography, and observe and record the colorimetric results for each concentration of sample. Based on the color development of the test line and control line, determine whether the test result is positive or negative. Based on the results of the three detection methods, the lowest template concentration that can reliably detect a positive signal was taken as the detection limit of this method, thereby determining the sensitivity of the RPA-CRISPR / Cas12a method to the drug resistance mutation site of Mycoplasma bovis A2058G.

[0099] 2. Test Results The pMB-R template was serially diluted 10-fold to obtain concentrations ranging from 1.8 × 10⁻⁶. 8 A series of gradient samples up to 1.8 × 10 copies / μL were subjected to RPA amplification and CRISPR / Cas12a detection under the optimal reaction conditions determined in the previous optimization. The results were interpreted using three methods: real-time fluorescence curves, endpoint fluorescence observation under ultraviolet light, and test strip detection. The results are as follows: Figure 16 .

[0100] Figure 16 A represents the real-time fluorescence curve changes for samples at each concentration gradient. The results show that at a concentration of 10... 8 Up to 10 3 The fluorescence curves of copies / μL all increased significantly with reaction time, showing typical positive amplification curves; while 10 2 The fluorescence curves of the copies / μL, 10 copies / μL, and negative control tubes remained flat without significant fluctuations. This result indicates that the detection limit of this method under real-time fluorescence detection is 10.3 copies / μL.

[0101] Figure 16 B shows the endpoint fluorescence images of each reaction tube taken under a multi-functional developing instrument after the reaction is complete. Visible to the naked eye, at a concentration of 10... 8 Up to 10 3 The reaction tubes of copies / μL all showed varying degrees of green fluorescence signal, among which 10 copies / μL... 3 Significant fluorescence can still be observed even at copies / μL; while at 10 2 No fluorescence reaction was observed at 10 copies / μL or 10 copies / μL. Figure 16 The analysis results of the endpoint fluorescence value in C were consistent with the real-time fluorescence detection results, further confirming that the detection limit of this method is 10. 3 copies / μL.

[0102] Figure 16 D represents the test strip results for each concentration gradient sample. The results show that the sample concentration is 10... 8 Up to 10 4 The T line on the test strip with copies / μL completely disappeared, indicating a strong positive result; the sample concentration was 10. 3 When the sample concentration is 10 copies / μL, the T line on the test strip shows weak color development, indicating a weak negative result; 3 The T line on the test strip showed clear color up to 10 copies / μL, indicating a negative result. Based on the above results, the detection limit of the test strip method is 1.8 × 10⁻⁶. 4 copies / μL.

[0103] In summary, the RPA-CRISPR / Cas12a detection method established in this study exhibits good detection sensitivity for the A2058G drug resistance mutation site in bovine mycoplasma. The detection limit based on real-time fluorescence detection and endpoint fluorescence observation is 1.8 × 10⁻⁶. 3 The detection limit is 1.8 × 10⁻⁶ copies / μL, while the detection limit based on visual interpretation of the test strip is 1.8 × 10⁻⁶. 4 The two detection methods differ in sensitivity, but both can meet the detection requirements of drug resistance mutation sites in clinical samples. In practical applications, the appropriate interpretation method can be selected based on the detection conditions and requirements.

[0104] Example 9: RPA-CRISPR / Cas12a System Specificity Test 1. Test Methods To verify the specificity of the two RPA-CRISPR / Cas12a detection methods established above for detecting the macrolide resistance mutation site (A2058G) of Mycoplasma bovis, a drug-resistant strain of Mycoplasma bovis (Mb1) and related pathogens were selected for specificity testing.

[0105] The control strains used in this experiment included: *Proteus mirabilis* (accession number: ATCC 49565), *Mannial lysate* (accession number: ATCC 29696), *Pasteurella multocida* (accession number: ATCC 9656), *Salmonella* (accession number: CMCC 50071), *Staphylococcus aureus* (accession number: CCTCC AB2013186), and *Klebsiella pneumoniae* (accession number: ATCC 700603), and a negative control was also included. Genomic DNA was extracted from each strain, and RPA amplification and CRISPR-Cas12a detection were performed sequentially according to the previously established optimal reaction system and primers.

[0106] The result interpretation method is based on the operation in Example 8. If only the Mycoplasma bovis drug-resistant strain Mb1 sample (isolated in Example 1) shows a positive signal, while other related pathogen samples and negative controls show no positive signal, it indicates that the method has good specificity and can accurately identify the Mycoplasma bovis A2058G drug-resistant mutation site without cross-reacting with other pathogens.

[0107] 2. Test Results The drug-resistant Mycoplasma bovis strain Mb1 and six other related pathogens were used as experimental strains for detection using the RPA-CRISPR / Cas12a system. The detection results were comprehensively interpreted using three methods: real-time fluorescence curve, endpoint fluorescence under ultraviolet light, and color development on the test strip. The results are as follows: Figure 17 As shown. Figure 17 A shows the changes in real-time fluorescence curves for each bacterial strain. The results showed that only the fluorescence curve of Mb1 increased significantly with the passage of reaction time; while the fluorescence curves of the other six pathogenic bacteria and the negative control remained flat without significant increase. Figure 17 B shows the endpoint fluorescence of each reaction tube as captured by a multi-functional developing instrument after the reaction. Only tube Mb1 showed a bright green fluorescence signal, while the other tubes showed no obvious fluorescence, consistent with the real-time fluorescence detection results. Figure 17 D represents the test results of the test strips corresponding to each strain sample. The results show that only test strip No. 1 (Mb1) showed a positive result because the C line was colored while the T line was not colored; the other test strips showed a clear T line and a normal C line, which were all negative results.

[0108] In summary, the results from the three detection methods—real-time fluorescence curve, endpoint fluorescence observation, and test strip color development—are highly consistent, all indicating that the RPA-CRISPR / Cas12a detection method established in this experiment only produces a specific recognition signal for drug-resistant Mycoplasma bovis strains, while showing no cross-reactivity with the other six related pathogens. This result demonstrates that the established detection method has good specificity and can accurately identify the drug-resistant mutation site of Mycoplasma bovis A2058G, providing a reliable guarantee of specificity for subsequent clinical sample testing.

[0109] Example 10: Application of RPA-CRISPR / Cas12a detection method 1. Test Methods To evaluate the effectiveness of the established RPA-CRISPR / Cas12a detection method in sample detection, this study used 32 isolated and identified Mycoplasma bovis strains as subjects. Based on the optimal conditions optimized in Examples 4 and 6-7, RPA amplification and detection using two CRISPR / Cas12a systems were performed on the samples. The samples tested included 13 drug-resistant strains (Mb1, Mb7, Mb12, Mb13, Mb14, Mb15, Mb16, Mb17, Mb24, Mb25, Mb26, Mb28, Mb32) and 19 sensitive strains (Mb2, Mb3, Mb4, Mb5, Mb6, Mb8, Mb9, Mb10, Mb11, Mb18, Mb19, Mb20, Mb21, Mb22, Mb23, Mb27, Mb29, Mb30, Mb31). Each group of samples underwent RPA-CRISPR / Cas12a reaction and test strip testing, with a negative control included. Results were interpreted according to the procedures described in Example 8. The detection results were compared with known strain information to calculate the detection rate, thereby evaluating the reliability of the fluorescence detection method and the test strip testing method in sample detection.

[0110] 2. Test Results The test results of 32 bovine mycoplasma samples are as follows Figure 18 As shown. Figure 18 The results of the drug-resistant strain detection of A showed that the real-time fluorescence curves of all 13 drug-resistant strains showed an upward trend, and obvious green fluorescence signals were observed in all reaction tubes. Figure 18 The results of the detection of sensitive strains B showed that the real-time fluorescence curves of all 19 sensitive strains remained flat without significant fluctuations; no fluorescence signal was observed in any reaction tubes after the reaction was completed. The results indicate that the results obtained from the RPA-CRISPR / Cas12a detection of 32 bovine mycoplasma clinical samples were completely consistent with the drug susceptibility test results, with a detection rate of 100%.

[0111] The test strip results for drug-resistant and susceptible strains are as follows: Figure 19As shown in the image, the C line of the test strip corresponding to the drug-resistant strain Mb14 was clearly visible, but the T line was faintly visible, resulting in a weak negative result, making it impossible to distinguish its drug-resistant phenotype. Analysis suggests that the low recognition efficiency of crRNA and target sequence in this reaction system led to insufficient cleavage of the reporter probe, resulting in the faint T line. All other drug-resistant strains showed typical positive results: the C line was clearly visible, and the T line was completely eliminated. The test strips corresponding to sensitive strains all showed negative results: both the C and T lines were clearly visible. The results of the test strips for sensitive strains were highly consistent with the fluorescence detection results. However, for drug-resistant strains, the results for Mb14 differed from the fluorescence detection results, while the results for the other drug-resistant strains were consistent. In summary, the detection rate of the test strip method was 96.8%.

[0112] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A primer pair, characterized in that, This includes the upstream primer PAM-F6 as shown in SEQ ID NO.12 and the downstream primer PAM-R as shown in SEQ ID NO.

6.

2. A crRNA, characterized in that, Its sequence is shown in SEQ ID NO.

15.

3. A reagent kit, characterized in that, This includes RPA primer pairs for amplifying gene mutation sites A2058G, specific crRNA targeting gene mutation sites A2058G, and fluorescent reporter probes. The gene mutation site A2058G is located in the 23S rRNA of bovine mycoplasma ribosomes. rrnA The 2058th base in the V region of the gene, wherein the 2058th base is either A or G; The RPA primer pair used to amplify the gene mutation site A2058G includes the upstream primer PAM-F6 as shown in SEQ ID NO.12 and the downstream primer PAM-R as shown in SEQ ID NO.6; The sequence of the specific crRNA targeting the gene mutation site A2058G is shown in SEQ ID NO.

15.

4. The reagent kit according to claim 3, characterized in that, The nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO.

16.

5. The reagent kit according to claim 4, characterized in that, The fluorescent reporter probe is labeled with the fluorescent group FAM at its 5′ end and the quencher group BHQ1 at its 3′ end.

6. A method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a, characterized in that, Includes the following steps: S1. Extract genomic DNA from bovine mycoplasma samples; S2. Using the genomic DNA extracted in step S1 as a template, RPA amplification is performed using the upstream primer PAM-F6 shown in SEQ ID NO.12 and the downstream primer PAM-R shown in SEQ ID NO.6 to obtain the RPA amplification product. S3. The RPA amplification product obtained in step S2 is detected using a CRISPR / Cas12a system including crRNA6 as shown in SEQ ID NO.15 and ssDNA reporter as shown in SEQ ID NO.

16. The detection results are obtained using a fluorescence signal detector or chromatography strip.

7. The method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a according to claim 6, characterized in that, The RPA amplification reaction conditions were: constant temperature reaction at 39℃ for 20 min.

8. The method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a according to claim 6, characterized in that, The reaction system of the CRISPR / Cas12a system includes: 2 μL of 10×Reaction Buffer, 1 μL of Cas12a protein, 0.6 μL of ssDNA reporter, 1 μL of RPA amplification product, and DEPC water to a final volume of 20 μL.

9. The method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a according to claim 8, characterized in that, When obtaining the detection results using a fluorescence signal detector, the concentration of ssDNA reporter in the CRISPR / Cas12a system reaction system was 200 nM, the concentration of Cas12a protein was 110 nM, and the concentration of crRNA6 was 50 nM; the reaction conditions for step S3 were: constant temperature reaction at 37℃ for 1 h.

10. The method for detecting single-base drug resistance mutations in bovine mycoplasma based on RPA-CRISPR / Cas12a according to claim 8, characterized in that, When obtaining the test results using the chromatography test strip, the concentration of ssDNA reporter is 60 nM; the reaction conditions for step S3 are: constant temperature reaction at 37℃ for 40 min.

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