SNP (Single Nucleotide Polymorphism) site for distinguishing Mycoplasma synoviae MS-H vaccine strain and wild strain, kit and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an SNP (Single Nucleotide Polymorphism) site for distinguishing a mycoplasma synoviae MS-H vaccine strain and a wild strain, a kit and application, and target genes meeting detection requirements are screened out by carrying out bioinformatics comparative analysis on whole genomes of MS-H and parent strains thereof. A specific primer and an MGB probe are designed for a specific conserved gene sequence ktrB gene for the first time, and the double qPCR kit for distinguishing the MS-H vaccine strain and the wild strain is established. In-vitro experiments prove that the kit disclosed by the invention is simple to operate, high in sensitivity, strong in specificity and good in repeatability. The screened gene loci and the established detection kit have the advantages that the defect that at present, common genes are prone to reverse mutation is overcome, the method can be used for clinical rapid diagnosis of MS vaccine strains and wild strains, the detection accuracy is improved, population purification is promoted, and certain commercial value is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a SNP site, a kit and an application thereof for distinguishing a Mycoplasma synoviae MS-H vaccine strain from a wild strain. Background Art

[0002] Mycoplasma synoviae (MS) can cause bursitis and air sac inflammation in poultry, leading to a decrease in egg production and quality, a decrease in the hatchability of eggs, an increase in the culling rate of chicks, and an impact on the growth and development of broilers. MS exists in chickens for a long time and is often mixed with other pathogens, such as Newcastle disease virus and Escherichia coli, causing huge economic losses to the poultry industry. Therefore, the prevention, control and purification of MS has always been a problem that has plagued the poultry industry, and is also the focus of research in modern intensive poultry farming.

[0003] Currently, the live vaccine used to prevent MS infection is mainly the Australian attenuated live vaccine strain MS-H. The application of live vaccines can, on the one hand, produce a competitive exclusion effect on wild strain infection by colonization in the upper respiratory tract, and on the other hand, it can also produce an effective mucosal immune response. The application of this vaccine can significantly alleviate clinical symptoms, but it cannot completely avoid infection with wild strains. In addition, since MS can be transmitted horizontally and vertically, it interferes with the results of serological monitoring, making it difficult to distinguish between immunized chickens and wild-type infected chickens. Therefore, in order to monitor the infection of chickens and implement MS control and eradication plans, it is urgent to establish a more accurate detection method to distinguish infected animals from vaccinated animals (Differentiation of infected from vaccinated animals, DIVA).

[0004] The differential diagnosis methods for MS include serological methods and molecular biological tests. Serological diagnosis has nonspecific reactions and cross reactions, and is prone to false positive results. Molecular biology technology is currently a widely used detection method that can quickly detect MS in samples and greatly improve detection efficiency. However, conventional PCR methods have low specificity, and certain components in complex samples have an inhibitory effect on PCR amplification, which is prone to false positives, and there is cross contamination in gel electrophoresis. The development of realtime fluorescence quantitative PCR (qPCR) circumvents these problems, using completely closed tube detection to avoid cross contamination, and the sensitivity is significantly higher than that of ordinary 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 ynoviae 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, specificity duplex 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 established qPCR methods based on SNP mutation sites in the obg gene to differentiate between MS vaccine strains and wild strains. However, studies have found that the obg and oppF genotypes of some MS-H re-isolated strains can be restored to wild strains, so 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 strains, and establishing an efficient and accurate detection method based on new sites, is of great significance for the purification of mycoplasma-infected chickens. Summary of the invention

[0006] The purpose of the present invention is to provide a SNP site, a kit and an application for distinguishing MS-H vaccine strains from wild strains of Mycoplasma synoviae, which provides a reliable identification target for the establishment of a detection method for distinguishing MS-H strains from wild strains, greatly improves the accuracy and credibility of clinical detection, and makes up for the shortcomings of existing detection methods.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a SNP site for distinguishing the MS-H vaccine strain of Mycoplasma synoviae from the wild strain, and the SNP sites include 21, as shown in the following table:

[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 strain of Mycoplasma synoviae. The specific primer and probe combination is designed based on the ktrB site with a gene number of MSH_02655. The specific primers include specific primers MS-ktrB-F and MS-ktrB-R, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The probe combination includes a vaccine strain MS-H specific probe MS-ktrB-VP and an MS wild strain specific probe MS-ktrB-WP, whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

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

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

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

[0015] Furthermore, the two positive standards are respectively 822 Vaccine strain MS-H and wild strain MS WVU including mutation sites 1853 The plasmid containing the ktrB gene fragment.

[0016] The present invention also provides a method for distinguishing the Mycoplasma synoviae vaccine strain MS-H from wild strains, the method comprising the step of using the kit to detect a sample to be tested.

[0017] Further, the method specifically includes the following steps:

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

[0019] (2) The positive standard plasmid was transferred from 10 7 Copies / μL to 10 0 The copies / μL gradient dilution was used as a template, and fluorescent quantitative PCR amplification was performed with the specific primer and probe combination to establish a standard curve;

[0020] (3) Using the genomic DNA extracted in step (1) as a template, fluorescent quantitative PCR amplification is performed using the specific primer and probe combination.

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

[0022] Furthermore, the reaction system for the fluorescent quantitative PCR amplification is 20 μL, containing 2×AceQ Universal U+Probe Master 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, and sterile ultrapure water 5.6 μL; the reaction procedure for the fluorescent quantitative PCR amplification is: contamination 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, and 45 cycles.

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

[0024] Beneficial effects:

[0025] The present invention provides 21 SNP sites that can be used to distinguish MS temperature-sensitive vaccine strains (MS-H strains) from wild strains. These SNP sites are not only highly conserved in all wild strains, but also stably exist in MS-H and its re-isolated strains, providing reliable identification targets for the establishment of a detection method for distinguishing MS-H strains from wild strains.

[0026] The present invention is the first to design specific primers and MGB probes based on the specific conservative gene sequence ktrB gene to establish a detection kit. The use of the ktrB gene site can eliminate detection errors caused by reverse mutations of existing detection targets, greatly improve the accuracy and credibility of clinical detection, and make up for the shortcomings of existing detection methods.

[0027] The kit provided by the present invention optimizes the annealing temperature, primer set and probe concentration ratio used in the kit, and performs sensitivity and specificity verification and clinical sample detection. In vitro experiments have shown that the kit of the present invention has high sensitivity, strong specificity and good repeatability, can be used for rapid differentiation between MS-H vaccine strains and wild-type infection strains, is suitable for early detection of clinical infections, and can also be used for identification of isolated strains. As a new and accurate detection method, the present invention provides a new idea for the monitoring, purification and laboratory diagnosis of synoviae Mycoplasma in samples from farms and breeders, which is conducive to timely treatment and prevention of diseases and shortens population purification time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The ktrB gene primer and probe sequence design diagram of Example 2 of the present invention;

[0030] Figure 2 It is an amplification curve diagram of the FAM fluorescence signal (vaccine) and the 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 gradient diluted wild strain standard in Example 3 of the present invention;

[0032] Figure 4 The standard curve corresponding to the gradient diluted wild strain standard in Example 3 of the present invention;

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

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

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

[0036] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

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

[0040] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources.

[0041] Example 1 Screening Method for Potential Detection Sites of MS-H Vaccine Strains and Wild Strains

[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. All SNP sites were initially screened using MAUVE software, and then analyzed for conservation and specificity using NCBI's BLAST analysis. The 21 conservative and specific target sites finally obtained all met the following requirements: stable SNP mutations in MS-H and its parent strains, SNP sites in all wild strains were consistent with the parent strains, and SNP sites in MS-H and all its re-isolated strains had consistent mutations that were different from wild strains. As shown in Table 1.

[0043] Table 1 Target gene loci for distinguishing MS-H vaccine strains from wild strains of Mycoplasma synoviae

[0044]

[0045]

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

[0047] 1. Primer and probe design

[0048] The sequences in Table 1 were aligned using MEGA11 (the results are shown in Figure 1 As shown), upstream and downstream primers were designed in the conservative segment containing the mutation site. Then, according to the differences in SNP sites, vaccine strain-specific probes and wild strain-specific probes were designed. The vaccine strain probe is labeled with a fluorescent reporter group FAM at the 5' end and a fluorescent quencher group MGB at the 3' end; the wild strain probe is labeled with a fluorescent reporter group CY5 at the 5' end and a fluorescent quencher group MGB at the 3' end. The primers and probes are synthesized by Shanghai Ruimian Biotechnology Co., Ltd., China. After experimental verification, the ktrB gene with good specificity and sensitivity was selected for subsequent experiments. The upstream and downstream primers of the ktrB gene are named MS-ktrB-F (SEQ ID NO.1) and MS-ktrB-R (SEQID NO.2), respectively; the vaccine strain and wild strain-specific probes are named MS-ktrB-VP (SEQ ID NO.3) and MS-ktrB-WP (SEQ ID NO.4), respectively, and the sequences are shown in Table 2.

[0049] Table 2 Primer and probe sequences

[0050]

[0051] 2. Genome template preparation

[0052] ResuscitationMS-H, MS WVU 1853 , MS clinical isolates, and non-MS avian strains were cultured and expanded. The supernatant was discarded after centrifugation. The precipitate was resuspended in sterile enzyme-free water, oscillated and mixed, and crudely extracted in a 100°C metal bath for 20 minutes. The supernatant after centrifugation was the genomic DNA, which was stored at -20°C for later use.

[0053] 3. Perform fluorescence quantitative PCR reaction

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

[0055] Result determination method: observe whether there is a characteristic S curve in the fluorescence quantitative PCR amplification. When the CY5 signal is detected and the characteristic S curve is presented, it is determined to be the MS wild strain. When the FAM signal is detected and the characteristic S curve is presented, it is determined to be the MS-H vaccine strain. On the basis of the 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, by trying different annealing temperatures (58, 60, 62°C), it was found that when the annealing temperature was 60°C, the non-specific signal was the lowest and the Ct value was relatively low. Then, based on the three-factor five-level orthogonal experiment, 25 groups of reaction systems were designed to optimize the concentrations of primers and probes. The final reaction conditions are as follows.

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

[0059] Example 3 Evaluation of the real-time fluorescence quantitative PCR method for distinguishing MS-H vaccine strains from wild strains based on new SNP sites

[0060] 1. Specificity evaluation

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

[0062] The MS vaccine strain (MS-H strain) was purchased from Australian Bioresources; the MS wild-type strain (WVU 1853 strain) (Accession No.: CVCC385), Mycoplasma gallisepticum (MG) F strain (Accession No.: CVCC1652) are preserved in our laboratory; MG 08F8, MG 8-15, MS SH1 and MS HB1 are clinical strains of avian mycoplasma preserved in our laboratory. 4 clinical isolates of Escherichia coli (Wang Yao, Zhang Yaodong, Yi Zhengfei, et al. Molecular epidemiological investigation of serotypes, evolutionary groups and virulence genes of avian pathogenic Escherichia coli [J]. Chinese Journal of Veterinary Science, 2020, 50(09): 1159-1166.); CT-1, CY-1, and LH-4 of Clostridium perfringens from chicken (Wang Di, Wang Xinyu, Guo Weiqi, et al. Isolation, identification and biological characteristics analysis of 16 strains of Clostridium perfringens from chicken in Jiangsu and Zhejiang regions [J]. Chinese Journal of Zooinfectious Diseases: 1-8.); SA01 isolate of Staphylococcus aureus and SP001 isolate of Salmonella pullorum (Zhang Beibei, Zhang Yaodong, Zhu Hong, et al. Analysis of the killing effect of hypobromous acid on common pathogenic bacteria of poultry at the scene of Hailuojing [J]. Chinese Journal of Zooinfectious Diseases, 2024, 32(03): 42-49.); Salmonella 2 clinical isolates (Xin Suhua, Tao Chenglin, Zhang Yaodong, et al. Isolation, identification and drug resistance analysis of Salmonella in chicken embryos in Guangxi [J]. Chinese Journal of Zooinfectious 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 Zooinfectious Diseases, 2019, 27(01): 49-54.); avian Proteus mirabilis (Wang Zhiyang, Peng Haoheng, Guo Weiqi, et al. Isolation, identification and drug resistance analysis of Proteus mirabilis producing NDM-1 type carbapenemase [J]. Chinese Journal of Veterinary Science, 2023, 53(12): 1554-1562.); Enterococcus faecalis Em1, Gallibacillus gallinulina Ga1, Riemerella anatipestifer SY-2, and Pasteurella hd are avian clinical strains recently isolated by our laboratory. The genomic DNA of the above 23 strains was extracted and the real-time qPCR test was performed under the optimized conditions to verify the specificity of this fluorescence quantitative method. Figure 2 .

[0063] Using the genomic DNA of the MS-H vaccine strain as a template, only the FAM fluorescence signal S curve can be detected, but no CY5 fluorescence signal S curve can be detected; using the genomic DNA of all MS wild strains as a template, only the CY5 fluorescence signal S curve can be detected, but no FAM fluorescence signal S curve can be detected; using non-MS genomic DNA as a template, both probe signals cannot be detected, and there is no S curve. The above results show that the real-time fluorescence quantitative PCR method for distinguishing MS-H vaccine strains from wild strains established by the present invention has good specificity, can distinguish MS infection 100%, and specifically identify MS-H vaccine strain infection.

[0064] 2. Standard curve establishment and sensitivity evaluation

[0065] The dual fluorescence quantitative PCR method established in Example 2 was used to prepare standards and establish a standard curve:

[0066] Primers ktrB-F (SEQ ID NO.5) and ktrB-R (SEQ ID NO.6) were used to isolate the wild-type MS strain WVU 1853 The genomic DNA of the vaccine strain (GenBank accession number: CP011096.1) and the MS-H vaccine strain (GenBank accession number: CP021129.1) were used as templates for PCR amplification, and the gel-recovered products were cloned into the PMD18-T vector (Takara Biotechnology Co., Ltd., catalog number: 6011) to obtain the recombinant plasmids of the vaccine strain and the wild strain. The recombinant plasmids were transformed into DH5α competent cells for culture, and the positive colonies with successful construction were selected for plasmid extraction and sent for sequencing. The concentration was measured with a spectrophotometer, and the copy number was calculated based on the concentration.

[0067] The plasmid was transferred from 10 7 copies / μL up to 10 0 A 10-fold serial dilution of 10 copies / μL was used as a template to establish the standard curve of the real-time qPCR method. Real-time fluorescence quantitative PCR amplification was performed according to the optimal conditions. The amplification curve of the wild strain standard and the drawn standard curve are shown in Figure 3 and Figure 4 The amplification curve of the MS-H vaccine strain standard and the drawn standard curve are shown in Figure 5 and Figure 6 As can be seen from the figure, the detection sensitivity of the real-time fluorescence quantitative PCR method for the two positive standards of MS-H vaccine and wild-type virus can reach 6.3 copies and 14.4 copies respectively. Through analysis, the standard curves of fluorescence quantitative PCR for detecting MS-H vaccine strains and MS wild-type virus strains have a good linear relationship, and the correlation coefficient (R 2 ) was 0.9992, and the correlation coefficient (R 2 ) 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 fluorescence quantitative 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-batch and inter-batch parallel tests. The intra-batch parallel test method is to repeat each sample three times in one PCR test; the inter-batch parallel test method is to test each sample three times in different batches at different times and under different operating conditions. The intra-batch and inter-batch coefficients of variation were calculated to analyze the repeatability of the test. The test results are shown in Tables 3 and 4, respectively. According to the test results, this method has strong repeatability, and the established dual fluorescence quantitative PCR detection method can distinguish the MS-H vaccine strain and the wild strain MS WVU with good repeatability. 1853 .

[0074] Table 3 Genome repetitiveness detection results

[0075]

[0076] Table 4 Standard repeatability test results

[0077]

[0078]

[0079] 4. Interference evaluation

[0080] The wild-type MS strain (WVU 1853) genomic DNA was diluted in a gradient of 1:10, 1:50, 1:100, 1:500, 1:1000, and 1:5000, and each dilution was mixed with equal amounts of vaccine strain (MS-H) genomic DNA and control (sterile ultrapure water), and the above method was used for detection. The MS-H vaccine strain genomic DNA was diluted to the same dilution in the same way, and then the interference group and control group were tested. Real-time fluorescence quantitative PCR amplification was performed according to the preferred conditions, and the test results are shown in Table 5. According to the test results, it can be seen that the method has a strong anti-interference ability, and can still accurately achieve the detection purpose in the presence of interference, and can be used to detect wild strains and vaccine strains separately when they exist at the same time.

[0081] Table 5 Interference test results for detection of wild strains and vaccine strains

[0082]

[0083] Example 4 Clinical validation of the real-time fluorescence quantitative PCR method for distinguishing MS-H vaccine strains from wild strains based on the ktrB gene

[0084] 1. Throat swab contaminated samples

[0085] A total of 33 throat swabs were collected from SPF chickens and chickens infected with Salmonella, soaked in 300 μL of PBS buffer, added with 30 μL of bacterial solution in the logarithmic phase of culture, and DNA was extracted using an oral swab genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., product number: DP362). qPCR amplification was performed according to the preferred conditions to determine the results. A total of 30 MS-H contaminated positive samples and 3 negative samples were included. 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 shows that this method can completely distinguish between positive and negative MS samples and can be used for differential diagnosis during clinical infection.

[0086] Table 6 Results of throat swab contamination sample testing

[0087]

[0088] 2. Tissue contamination samples

[0089] Fifteen lung samples were collected from SPF chickens and chickens infected with Salmonella. Different concentrations of bacterial solution were added to the experimental group, and PBS was used instead in the control group. DNA was extracted using a tissue genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., product number: DP304). qPCR amplification was performed according to the preferred conditions to determine the results. A total of 13 positive samples contaminated with wild-type MS strains and 2 negative samples were included. The test results are shown in Table 7. According to the test results, the results of fluorescence quantitative PCR identification were completely consistent with the expected results, with an accuracy rate of 100%. 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 Test results of tissue contamination samples

[0091]

[0092]

[0093] In summary, the traditional detection method of MS is serological detection, but because MS can be transmitted horizontally and vertically, the serological detection results are inaccurate. In recent years, molecular biology methods have been widely used in MS detection. Compared with ordinary PCR detection methods, qPCR can significantly improve accuracy and sensitivity, and can achieve full-process monitoring and shorten detection time, and has a wide range of application value. It is reported that the commonly used MS detection genes are obg and oppF, both of which have the possibility of reverse mutation. Therefore, in order to establish a more accurate and reliable detection method, the present invention performs bioinformatics comparison analysis on the whole genome of MS-H and its parent strain, and screens out 21 SNP sites with detection potential. In addition, the present invention is the first to use the ktrB gene to establish a dual fluorescence PCR method based on TaqMan-MGB probe, which is simple to operate, has strong anti-interference, strong specificity, high sensitivity and good stability.

[0094] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A SNP site for distinguishing Mycoplasma synoviae MS-H vaccine strain from wild strains, characterized in that: The SNP sites include 21, as shown in the following table:

2. A specific primer and probe combination that can be used to distinguish between the MS-H vaccine strain and the wild strain of Mycoplasma synoviae, characterized in that: The specific primer and probe combination is designed based on the ktrB site with the gene number MSH_02655 in claim 1, the specific primers include specific primers MS-ktrB-F and MS-ktrB-R whose sequences are 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 MS wild strain specific probe MS-ktrB-WP whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

3. The specific primer and probe combination according to claim 2, characterized in that: The vaccine strain MS-H specific probe MS-ktrB-VP is labeled with a FAM reporter group at its 5' end and a MGB quencher group at its 3' end, and the MS wild strain specific probe MS-ktrB-WP is labeled with a CY5 reporter group at its 5' end and a MGB quencher group at its 3' end.

4. A kit prepared using the primer combination according to any one of claims 2 to 3, characterized in that: The kit comprises the specific primer and probe combination according to claim 2.

5. The kit according to claim 4, characterized in that The kit also includes 2 positive standards, 2×AceQ Universal U+Probe Master Mix V2 and sterile ultrapure water.

6. The kit according to claim 5, characterized in that The two positive standards contain the key ktrB 822 Vaccine strain MS-H and wild strain MS WVU including mutation sites 1853 The plasmid containing the ktrB gene fragment.

7. A method for distinguishing Mycoplasma synoviae vaccine strain MS-H from wild strains, characterized in that: The method comprises the step of detecting a sample to be tested using the kit according to any one of claims 4 to 6.

8. The method for distinguishing the Mycoplasma synoviae vaccine strain MS-H from the wild strain according to claim 7, characterized in that: The specific steps include: (1) Extracting genomic DNA from the sample to be tested; (2) The positive standard plasmid was transferred from 10 7 Copies / μL to 10 0 The copies / μL gradient dilution is used as a template, and fluorescent quantitative PCR amplification is performed with the specific primer and probe combination described in claim 2 to establish a standard curve; (3) Using the genomic DNA extracted in step (1) as a template, fluorescent quantitative PCR amplification is performed using the specific primer and probe combination described in claim 2. (4) Observe whether the fluorescence quantitative PCR amplification in step (3) has a characteristic S curve. When a CY5 signal is detected and a characteristic S curve is presented, it is determined to be an MS wild strain. When a FAM signal is detected and a characteristic S curve is presented, it is determined to be an MS-H vaccine strain. On the basis that the curve is normal, quantify the strain DNA content in the sample to be tested according to the standard curve established in step (2).

9. The method for distinguishing the Mycoplasma synoviae vaccine strain MS-H from the wild strain according to claim 8, characterized in that: The reaction system for the fluorescent quantitative PCR amplification is 20 μL, containing 2×AceQ Universal U+Probe MasterMix 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, and sterile ultrapure water 5.6 μL; the reaction procedure for the fluorescent quantitative PCR amplification is: contamination 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, and 45 cycles.

10. Use of the SNP site according to claim 1, or the specific primer and probe combination according to any one of claims 2-3, or the kit according to any one of claims 4-6 in distinguishing between the MS-H vaccine strain and the wild strain of Mycoplasma synoviae, characterized in that: The main basis for distinguishing the MS-H vaccine strain of Mycoplasma synoviae from the wild strain is that there are stable SNP mutations in MS-H and its parent strains, the SNP sites in all wild strains are consistent with the mutations of the parent strains, and the SNP sites in MS-H and all its re-isolated strains have consistent mutations that are different from the wild strains.

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