SNP site for distinguishing mycoplasma synoviae ms-h vaccine strain from wild strain, kit and application

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a SNP site for distinguishing between MS-H vaccine strains and wild strains of synovial bursa mycoplasma, a kit and application. ktrb The application first designs specific primers and MGB probes for specific conservative gene sequences, and establishes a double qPCR kit for distinguishing between MS-H vaccine strains and wild strains. Experiments in vitro prove that the kit is simple to operate, high in sensitivity, strong in specificity, and good in repeatability. The gene site and the detection kit have the advantages of avoiding the deficiency that the commonly used genes are prone to revert mutation, can be used for clinical rapid diagnosis of MS vaccine strains and wild strains, improve the accuracy of detection, promote population purification, and have certain commercial value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an SNP site, kit, and application for distinguishing between MS-H vaccine strains and wild-type strains of Mycoplasma synovitis. Background Technology

[0002] Mycoplasma synoviae (MS) infection in poultry can cause synocasitis and air sacculitis, leading to decreased egg production and quality in laying hens, reduced hatchability of hatching eggs, increased culling rate of chicks, and negatively impacting the growth and development of broilers. MS is a persistent presence in chicken flocks and is often co-infected with other pathogens, such as Newcastle disease virus and Escherichia coli, causing significant economic losses to the poultry industry. Therefore, the prevention and eradication of MS has always been a challenging problem in the poultry industry and a key research focus in modern intensive poultry farming.

[0003] Currently, the main live vaccine used to prevent MS infection is the Australian attenuated live vaccine strain MS-H. The application of live vaccines can competitively exclude wild-type strains through colonization in the upper respiratory tract and can also generate an effective mucosal immune response. This vaccine can significantly reduce clinical symptoms, but it cannot completely prevent wild-type strain infection. Furthermore, because MS can spread horizontally and vertically, it interferes with serological monitoring results, making it difficult to distinguish between immunized flocks and wild-type infected flocks. Therefore, in order to monitor the infection status of chicken flocks and implement MS control and eradication programs, there is an urgent need to establish a more accurate detection method to differentiate between infected and vaccinated animals (Differentiation of infected from vaccinated animals, DIVA).

[0004] Differential diagnosis of MS includes serological methods and molecular biological assays. Serological diagnosis suffers from nonspecific reactions and cross-reactivity, easily leading to false positive results. Molecular biological techniques are currently the most widely used detection methods, enabling rapid detection of MS in samples and significantly improving detection efficiency. However, conventional PCR methods have low specificity, and certain components in complex samples can inhibit PCR amplification, easily producing false positives. Furthermore, gel electrophoresis can lead to cross-contamination. The development of real-time fluorescence quantitative PCR (qPCR) avoids these problems by using completely closed tube detection, preventing cross-contamination, and exhibiting significantly higher sensitivity than conventional PCR methods.

[0005] In recent years, SNP-based qPCR detection methods have been widely used. R. Dijkman et al. (Dijkman R, Feberwee A, Landman WJ M. Development, validation and wild-type evaluation of a quantitative real-time PCR able to differentiate between wild-type Mycoplasma synoviae and the MS-H-live vaccine strain[J]. Avian Pathology, 2017, 46(4):403-415.) and Liu Ruidong et al. (Liu R, Lin Q, Cai Q, et al. A novel high sensitive, specificityduplex enzyme-activated differentiating probes PCR method for the SNP detection and differentiation of MS-H vaccine strains from wild-type Mycoplasma synoviae strains[J]. Poult Sci, 2024, 103(8):103874.) have both established qPCR methods based on SNP mutation sites in the obg gene to differentiate MS vaccine strains from wild-type strains. However, studies have found that the obg and oppF genotypes of some MS-H resembling strains can revert to wild-type strains, thus the detection method based on this site has errors (Klose SM, Olaogun OM, Disint JF, et al. Genomic diversity of a globally used, live attenuated mycoplasma vaccine[J]. Microbiology spectrum, 2022, 10(6):e0284522.). Therefore, screening stable SNP sites that can distinguish between MS vaccine strains MS-H and wild-type strains, and establishing an efficient and accurate detection method based on the new sites, is of great significance for the eradication of mycoplasma infection in chicken flocks. Summary of the Invention

[0006] The purpose of this invention is to provide an SNP site, kit, and application for distinguishing between MS-H vaccine strains and wild-type strains of Mycoplasma synovitis. This provides a reliable identification target for establishing a detection method to differentiate between MS-H strains and wild-type strains, greatly improving the accuracy and reliability of clinical testing and making up for the shortcomings of existing detection methods.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a SNP site for distinguishing between Mycoplasma synovitis MS-H vaccine strains and wild-type strains. The SNP site includes 21 sites, as shown in the table below:

[0009]

[0010]

[0011] The present invention also provides a specific primer and probe combination that can be used to distinguish between the MS-H vaccine strain and the wild-type strain of Mycoplasma synovitis. The specific primer and probe combination is designed based on the ktrB site of gene number MSH_02655. The specific primers include specific primers MS-ktrB-F and MS-ktrB-R with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The probe combination includes MS-ktrB-VP, a vaccine strain-specific probe for MS-H, and MS wild-type strain-specific probe MS-ktrB-WP, with sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0012] Furthermore, the 5' end of the MS-H specific probe MS-ktrB-VP of the vaccine strain is labeled with a FAM reporter group and the 3' end is labeled with an MGB quencher group, and the 5' end of the MS wild-type specific probe MS-ktrB-WP is labeled with a CY5 reporter group and the 3' end is labeled with an MGB quencher group.

[0013] The present invention also provides a kit prepared using the primer composition described above, characterized in that the kit comprises the specific primer and probe combination described above.

[0014] Furthermore, the kit also includes two positive standards, 2×AceQ Universal U+ProbeMaster Mix V2, and sterile ultrapure water.

[0015] Furthermore, the two positive standards contain the key ktrB, respectively. 822 The vaccine strain MS-H and wild-type strain MS WVU, including the mutation site 1853 The plasmid containing the ktrB gene fragment.

[0016] The present invention also provides a method for distinguishing between the Mycoplasma synovitis vaccine strain MS-H and the wild-type strain, the method comprising the step of detecting the sample to be tested using the kit described herein.

[0017] Furthermore, the specific steps include:

[0018] (1) Extract genomic DNA from the sample to be tested;

[0019] (2) Change the positive standard quality particles from 10 7 Copy / μL to 10 0 The copy / μL serial dilution was used as a template, and the specific primer and probe combination was used for real-time PCR amplification to establish a standard curve.

[0020] (3) Using the genomic DNA extracted in step (1) as a template, perform real-time PCR amplification with the specific primer and probe combination described above.

[0021] (4) Observe whether there is a characteristic S curve in the fluorescence quantitative PCR amplification in step (3). When the CY5 signal is detected and a characteristic S curve is presented, it is determined to be the MS wild virus strain. When the FAM signal is detected and a characteristic S curve is presented, it is determined to be the MS-H vaccine strain. On the basis of normal curve, the DNA content of the strain in the sample to be tested is quantified according to the standard curve established in step (2).

[0022] Furthermore, the reaction system for the quantitative real-time PCR amplification is 20 μL, containing 10 μL of 2×AceQ Universal U+Probe Master Mix V2, 0.8 μL of 10 μM MS-ktrB-F, 0.8 μL of 10 μM MS-ktrB-R, 0.4 μL of 10 μM MS-ktrB-VP, 0.4 μL of 10 μM MS-ktrB-WP, 2 μL of template, and 5.6 μL of sterile ultrapure water; the reaction program for the quantitative real-time PCR amplification is: 37℃ contamination digestion for 2 min; 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s; 60℃ annealing for 35 s, for 45 cycles.

[0023] This invention also provides the application of the SNP site, the specific primer and probe combination, or the kit described herein in distinguishing between Mycoplasma synoviae MS-H vaccine strain and wild-type strain. The main basis for distinguishing between Mycoplasma synoviae MS-H vaccine strain and wild-type strain is that MS-H and its parent strain have stable SNP mutations, the SNP sites in all wild-type strains are consistent with the mutations in the parent strain, and the SNP sites in MS-H and all its reisolated strains have consistent mutations that are different from those in the wild-type strain.

[0024] Beneficial effects:

[0025] This invention provides 21 SNP sites that can be used to distinguish between MS temperature-sensitive vaccine strains (MS-H strains) and wild-type strains. These SNP sites are not only highly conserved in all wild-type strains, but also stably present in MS-H and its reisolated strains, providing reliable identification targets for the establishment of detection methods to distinguish between MS-H strains and wild-type strains.

[0026] This invention is the first to design specific primers and MGB probes based on the specific conserved gene sequence ktrB, and establish a detection kit. The use of the ktrB gene locus eliminates detection errors caused by reversion mutations at existing detection targets, greatly improving the accuracy and reliability of clinical testing and overcoming the shortcomings of existing detection methods.

[0027] The kit provided by this invention has been optimized for annealing temperature and primer-to-probe concentration ratio, and its sensitivity, specificity, and clinical sample detection have been verified. In vitro experiments have demonstrated that the kit exhibits high sensitivity, strong specificity, and good reproducibility, enabling rapid differentiation between MS-H vaccine strains and wild-type virus-infected strains. It is suitable for early detection of clinical infections and can also be used for the identification of isolated strains. As a novel and accurate detection method, this invention provides a new approach for the monitoring, purification, and laboratory diagnosis of Mycoplasma synoviae in samples from farms and livestock breeders, facilitating timely disease treatment and control, and shortening population purification time. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a diagram showing the primer and probe sequence design for the ktrB gene in Example 2 of the present invention;

[0030] Figure 2 This is an amplification curve of the FAM fluorescence signal (vaccine) and CY5 fluorescence signal (wild virus) collected in the specificity test of Example 3 of the present invention;

[0031] Figure 3 This is the amplification curve corresponding to the serially diluted wild-type virus standard in Example 3 of the present invention;

[0032] Figure 4 This is a standard curve diagram corresponding to the gradient dilution of the wild-type virus standard in Example 3 of the present invention;

[0033] Figure 5 This is the amplification curve corresponding to the serially diluted vaccine strain standard in Example 3 of the present invention;

[0034] Figure 6 This is a standard curve diagram corresponding to the graded diluted vaccine strain standard in Example 3 of the present invention. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Unless otherwise specified, all chemical reagents, biochemical reagents and materials used in this invention are commercially available.

[0041] Example 1: Screening method for potential detection sites of MS-H vaccine strain and wild-type strain

[0042] The whole genome sequences of the MS vaccine strain MS-H (GenBank accession number CP021129) and its parent strain MS86079 / 7NS (GenBank accession number CP029258) were downloaded from the NCBI database. Preliminary screening of all SNP sites was performed using MAUVE software, followed by conservation and specificity analysis using NCBI's BLAST analysis. The 21 conserved and specific target sites identified met the following requirements: stable SNP mutations in MS-H and its parent strains; SNP sites in all wild-type strains consistent with the parent strains; and SNP sites in MS-H and all reisolated strains exhibiting consistent mutations distinct from wild-type strains. (See Table 1).

[0043] Table 1. Target gene loci for differentiating between MS-H vaccine strains and wild-type strains of Mycoplasma synoviae.

[0044]

[0045]

[0046] Example 2: Establishment of a real-time quantitative PCR method for differentiating MS-H vaccine strains from wild-type strains based on the ktrB gene.

[0047] 1. Primer and probe design

[0048] Sequence comparisons were performed on the sites in Table 1 using MEGA11 (results are shown below). Figure 1 As shown in the figure, upstream and downstream primers were designed for the conserved segments containing the mutation sites. Then, vaccine-specific probes and wild-type-specific probes were designed to target the differences in SNP sites. The vaccine probes were labeled with the fluorescent reporter group FAM at the 5' end and the fluorescent quencher group MGB at the 3' end; the wild-type probes were labeled with the fluorescent reporter group CY5 at the 5' end and the fluorescent quencher group MGB at the 3' end. All primers and probes were synthesized by Shanghai Ruimian Biotechnology Co., Ltd., China. After experimental verification, the ktrB gene, which has good specificity and sensitivity, was selected for subsequent experiments. The upstream and downstream primers for the ktrB gene were named MS-ktrB-F (SEQ ID NO.1) and MS-ktrB-R (SEQ ID NO.2), respectively; the vaccine-specific and wild-type-specific probes were named MS-ktrB-VP (SEQ ID NO.3) and MS-ktrB-WP (SEQ ID NO.4), respectively, and their sequences are shown in Table 2.

[0049] Table 2 Primer and probe sequences

[0050]

[0051] 2. Genome template preparation

[0052] Recovery MS-H, MS WVU 1853 MS clinical isolates and non-MS avian strains were cultured and expanded. The supernatant was discarded by centrifugation, and the precipitate was resuspended in sterile enzyme-free water. The mixture was shaken and placed in a metal bath at 100°C for crude extraction for 20 min. The supernatant after centrifugation was the genomic DNA, which was stored at -20°C for later use.

[0053] 3. Perform quantitative real-time PCR reaction

[0054] Based on the MS-H and MS WVU obtained in step 2 1853 Using the DNA template and the specific primer and probe set designed and synthesized in this experiment, a dual real-time PCR reaction system was established according to the Vazyme (AceQ Universal U+Probe Master Mix V2) instructions. The above reaction system was used under predetermined real-time PCR reaction parameters: 37℃ digestion for 2 min; 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s; 60℃ annealing for 35 s; 45 cycles.

[0055] Result interpretation method: Observe whether the real-time PCR amplification has a characteristic S-curve. When a CY5 signal is detected and a characteristic S-curve is present, it is identified as an MS wild-type strain. When a FAM signal is detected and a characteristic S-curve is present, it is identified as an MS-H vaccine strain. Based on a normal curve, the DNA content of the strain in the sample to be tested is quantified according to the standard curve.

[0056] 4. Optimization of reaction conditions

[0057] First, using the basic reaction system, through experiments at different annealing temperatures (58, 60, 62℃), it was found that the nonspecific signal was lowest and the Ct value was relatively low when the annealing temperature was 60℃. Then, based on a three-factor, five-level orthogonal experiment, 25 reaction systems were designed to optimize the concentrations of primers and probes. The final reaction conditions are as follows.

[0058] The 20 μL system consisted of 10 μL of 2×AceQ Universal U+Probe Master Mix V2, 0.8 μL of 10 μM MS-ktrB-F, 0.8 μL of 10 μM MS-ktrB-R, 0.4 μL of 10 μM MS-ktrB-VP, 0.4 μL of 10 μM MS-ktrB-WP, 2 μL of template, and 5.6 μL of sterile ultrapure water. The reaction program was as follows: digestion at 37°C for 2 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s; annealing at 60°C for 35 s; 45 cycles.

[0059] Example 3: Evaluation of a real-time quantitative PCR method based on novel SNP sites for differentiating MS-H vaccine strains from wild-type strains.

[0060] 1. Specificity evaluation

[0061] The specificity of the dual-fluorescence quantitative PCR method established in Example 2 was evaluated:

[0062] MS vaccine strain (MS-H strain) was purchased from Australian Bioresources; MS wild-type strain (WVU) 1853 The strains of Mycoplasma gallisepticum (MG) F (accession number: CVCC385) and Mycoplasma gallisepticum (MG) F (accession number: CVCC1652) are preserved in this laboratory; MG 08F8, MG 8-15, MS SH1 and MS HB1 are clinical strains of Mycoplasma avianis preserved in this laboratory. Four clinical isolates of *Escherichia coli* (Wang Yao, Zhang Yaodong, Yi Zhengfei, et al. Molecular epidemiological investigation of serotypes, evolutionary groups and virulence genes of pathogenic *Escherichia coli* in birds [J]. *Chinese Journal of Veterinary Science*, 2020, 50(09): 1159-1166.); *Clostridium perfringens* CT-1, CY-1, LH-4 (Wang Di, Wang Xinyu, Guo Weiqi, et al. Isolation, identification and biological characteristics analysis of 16 strains of *Clostridium perfringens* from chickens in Jiangsu and Zhejiang provinces [J]. *Chinese Journal of Animal Infectious Diseases*: 1-8.); *Staphylococcus aureus* isolate SA01 and *Salmonella pullorum* isolate SP001 (Zhang Beibei, Zhang Yaodong, Zhu Hong, et al. Analysis of the killing effect of hypobromoic acid generated by phytosanitizer on common pathogenic bacteria in poultry [J]. *Chinese Journal of Animal Infectious Diseases*, 2024, 32(03): 42-49.); *Salmonella* Two clinical isolates (Xin Suhua, Tao Chenglin, Zhang Yaodong, et al. Isolation, identification and drug resistance analysis of Salmonella in chicken embryos in Guangxi region [J]. Chinese Journal of Animal Infectious Diseases, 2022, 30(03):64-70., Wu Xiaojun, Wang Shaohui, Yang Denghui, et al. Epidemiology and drug resistance analysis of Salmonella in East China [J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(01):49-54.); avian Proteus mirabilis (Wang Zhiyang, Peng Haoheng, Guo Weiqi, et al. Isolation, identification and drug resistance analysis of NDM-1 type carbapenemase-producing Proteus mirabilis [J]. Chinese Journal of Veterinary Science, 2023, 53(12):1554-1562.); Enterococcus viridissioides Em1, Agrobacterium galbana Ga1, Riemerella anatipestifer SY-2, and Pasteurella multocida HD were avian clinical strains recently isolated by our laboratory. Genomic DNA was extracted from the above 23 strains, and real-time qPCR was performed under optimized conditions to verify the specificity of this fluorescence quantitative method. Results are shown below. Figure 2 .

[0063] Using MS-H vaccine strain genomic DNA as a template, only the FAM fluorescence signal S-curve was detected, while the CY5 fluorescence signal S-curve was not detected. Using genomic DNA from all MS wild-type strains as a template, only the CY5 fluorescence signal S-curve was detected, while the FAM fluorescence signal S-curve was not detected. Using non-MS genomic DNA as a template, neither probe signal was detected, and no S-curve was observed. These results demonstrate that the real-time quantitative PCR method established in this invention for distinguishing between MS-H vaccine strains and wild-type strains has good specificity, can differentiate MS infection with 100% accuracy, and specifically identify MS-H vaccine strain infection.

[0064] 2. Establishment of standard curve and sensitivity evaluation

[0065] Using the dual-fluorescence quantitative PCR method established in Example 2, standards were prepared and a standard curve was established:

[0066] Using primers ktrB-F (SEQ ID NO. 5) and ktrB-R (SEQ ID NO. 6), the MS wild-type strain WVU was used. 1853 Using genomic DNA from the vaccine strain (GenBank accession number: CP011096.1) and the MS-H vaccine strain (GenBank accession number: CP021129.1) as templates, PCR amplification was performed. The gel-recovered products were cloned into the PMD18-T vector (Takara Bio Inc., catalog number: 6011) to obtain recombinant plasmids of the vaccine strain and wild-type strain. These were transformed into DH5α competent cells and cultured. Successfully constructed positive colonies were selected for plasmid extraction and sequencing. The concentration was measured using a spectrophotometer, and the copy number was calculated based on the concentration.

[0067] Plasmid from 10 7 copies / μL to 10 0 10-fold serial dilutions of copies / μL were used as templates to establish a standard curve for the real-time qPCR method. Real-time quantitative PCR amplification was performed under optimized conditions. The amplification curves of the wild-type virus standard and the plotted standard curves are shown below. Figure 3 and Figure 4 The amplification curves and standard curves of the MS-H vaccine strain standards are shown in [reference needed]. Figure 5 and Figure 6 As shown in the figure, the detection sensitivity of this real-time quantitative PCR method for the MS-H vaccine and wild-type virus positive standards reached 6.3 copies and 14.4 copies, respectively. Analysis revealed that the standard curves for both the MS-H vaccine strain and the MS wild-type virus strain showed good linearity, and the correlation coefficient (R²) for the MS-H standard was relatively low. 2 The correlation coefficient (R) of the MS wild-type strain standard was 0.9992. 2 The value is 0.9953.

[0068] The primer sequences for amplifying the ktrB standard are as follows:

[0069] ktrB-F: 5′-gagtaccaattgcaaaaactaaaaag-3′ (SEQ ID NO.5)

[0070] ktrB-R:5′-gataattctacttatcatcggtggaat-3′(SEQ ID NO.6)

[0071] 3. Repeatability evaluation

[0072] Taking the dual real-time PCR method established in Example 2 as an example, the repeatability of the detection method was evaluated:

[0073] Three samples of different concentrations were used as templates for intra-assay and inter-assay parallel experiments. Intra-assay parallel experiments involved three replicates of each sample in a single PCR test; inter-assay parallel experiments involved three separate batches of each sample tested at different times and under different operating conditions. Intra-assay and inter-assay coefficients of variation were calculated to analyze the reproducibility of the experiments. The results are shown in Tables 3 and 4. The results indicate that the method has strong reproducibility, and the established dual-fluorescence quantitative PCR detection method can reproducibly distinguish between the MS-H vaccine strain and the wild-type MS WVU strain. 1853 .

[0074] Table 3. Results of Genomic Repeatability Detection

[0075]

[0076] Table 4. Results of Repeatability Tests for Standard Samples

[0077]

[0078]

[0079] 4. Interference Evaluation

[0080] MS wild-type strain (WVU) 1853Genomic DNA was serially diluted at ratios of 1:10, 1:50, 1:100, 1:500, 1:1000, and 1:5000. Each dilution was mixed with an equal volume of vaccine strain (MS-H) genomic DNA and a control (sterile ultrapure water), and the above method was used for detection. The MS-H vaccine strain genomic DNA was then diluted to the same concentrations using the same method, and the interference group and control group were tested. Real-time quantitative PCR amplification was performed under optimized conditions, and the results are shown in Table 5. The results indicate that this method has strong anti-interference ability and can still accurately achieve the detection purpose even in the presence of interference. It can be used to detect wild-type and vaccine strains separately when both are present.

[0081] Table 5. Results of interference tests for wild-type and vaccine strains.

[0082]

[0083] Example 4: Clinical validation of a real-time quantitative PCR method based on the ktrB gene for differentiating MS-H vaccine strains from wild-type strains.

[0084] 1. Contaminated throat swab samples

[0085] Thirty-three pharyngeal swabs were collected from SPF chickens and chickens infected with Salmonella. The swabs were immersed in 300 μL of PBS buffer, and 30 μL of bacterial culture in the logarithmic growth phase was added. DNA was extracted using an oral swab genomic DNA extraction kit (Tiangen Biotech Co., Ltd., product number: DP362). qPCR amplification was performed under optimized conditions, and the results were determined. A total of 30 MS-H contamination-positive samples and 3 negative samples were identified. The test results are shown in Table 6. According to the test results, the qPCR identification results were completely consistent with the expected results, with an accuracy rate of 100%. This indicates that this method can completely distinguish between positive and negative MS samples and can be used for differential diagnosis in clinical infection processes.

[0086] Table 6. Detection results of contaminated throat swab samples

[0087]

[0088] 2. Organize contaminated samples

[0089] Fifteen lung samples were collected from SPF chickens and chickens infected with Salmonella. Different concentrations of bacterial suspension were added to the experimental groups, while PBS was used as a substitute for the control group. DNA was extracted using a tissue genomic DNA extraction kit (Tiangen Biotech Co., Ltd., product number: DP304). qPCR amplification was performed under optimized conditions, and the results were determined. A total of 13 samples were positive for MS wild-type virus contamination, and 2 samples were negative. The detection results are shown in Table 7. According to the detection results, the quantitative real-time PCR identification results were completely consistent with the expected results, with an accuracy rate of 100%. This indicates that this method can completely distinguish between positive and negative MS samples and can be used for differential diagnosis in clinical infection processes.

[0090] Table 7 Results of Tissue Contamination Sample Detection

[0091]

[0092]

[0093] In summary, traditional MS detection methods rely on serological testing. However, due to the horizontal and vertical propagation of MS, serological results are often inaccurate. In recent years, molecular biology methods have been widely applied to MS detection. Compared to conventional PCR methods, qPCR significantly improves accuracy and sensitivity, enabling continuous monitoring and shortening detection time, thus possessing broad application value. It has been reported that commonly used MS detection genes are obg and oppF, both of which have the potential for reversion mutations. Therefore, to establish a more accurate and reliable detection method, this invention performs bioinformatics alignment analysis on the whole genomes of MS-H and its parental strains, screening out 21 SNP sites with detection potential. Furthermore, this invention is the first to utilize the ktrB gene to establish a dual-fluorescence PCR method based on the TaqMan-MGB probe. This method is simple to operate, highly resistant to interference, highly specific, highly sensitive, and stable.

[0094] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for differentiating between Mycoplasma synoviae vaccine strain MS-H and field strains, characterized in that, The method comprises the following steps: (1) extracting genomic DNA of the sample to be detected; (2) The positive standard plasmid was diluted from 10 7 copies / μL to 10 0 copies / μL as a template, and a fluorescent quantitative PCR amplification was performed using primers ktrB-F with a sequence as shown in SEQ ID NO. 5 and primers ktrB-R with a sequence as shown in SEQ ID NO. 6, for establishing a standard curve; (3) performing fluorescent quantitative PCR amplification with the specific primer and probe combination and taking the genomic DNA extracted in step (1) as a template; (4) observing whether there is a characteristic S curve in the fluorescent quantitative PCR amplification in step (3), and determining that it is the MS wild strain when the CY5 signal is detected and a characteristic S curve is presented, and determining that it is the MS-H vaccine strain when the FAM signal is detected and a characteristic S curve is presented, and on the basis of the normal curve, quantifying the strain DNA content in the sample to be detected according to the standard curve established in step (2); The reaction system of the fluorescent quantitative PCR amplification is 20 μL, containing 2 × AceQ Universal U + ProbeMaster Mix V2 10 μL, 10 μM MS- ktrB - F 0.8 μL, 10 μM MS- ktrB - R 0.8 μL, 10 μM MS- ktrB - VP 0.4 μL, 10 μM MS- ktrB - WP 0.4 μL, template 2 μL, sterile ultrapure water 5.6 μL; the reaction program of the fluorescent quantitative PCR amplification is: 37℃ pollution digestion 2 min; 95℃ pre-denaturation 5 min; 95℃ denaturation 10 s, 60℃ annealing 35 s, 45 cycles; The specific primer and probe combination is based on the gene number MSH_02655 ktrB designed for the site, the specific primer includes the specific primer MS- ktrB -F and MS- ktrB -R, the sequence of which is shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively, and the probe combination includes the vaccine strain MS-H specific probe MS- ktrB -VP and the wild strain MS- ktrB -WP, the sequence of which is shown in SEQ ID NO. 3 and SEQ ID NO. 4 respectively. The vaccine strain MS-H specific probe MS- ktrB - VP is labeled with a FAM reporter group at the 5' end and a MGB quencher group at the 3' end, the MS wild strain specific probe MS- ktrB - WP is labeled with a CY5 reporter group at the 5' end and a MGB quencher group at the 3' end. The gene numbered MSH_02655 ktrB ktrB The specific information of the sites is shown as follows: 。

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