Mycoplasma gallisepticum and mycoplasma synoviae double qPCR primer group and detection method thereof

Abstract

The invention provides a double qPCR (quantitative polymerase chain reaction) primer combination of mycoplasma gallisepticum and mycoplasma synoviae. The double qPCR primer combination comprises specific primers and probes for detecting MG mgC2 genes of the mycoplasma gallisepticum and MS vlhA genes of the mycoplasma synoviae; a specific primer aiming at the MG mgC2 gene of the mycoplasma gallisepticum is SEQ ID NO.1-2, and a probe is MG-Taq; a specific primer for the Mycoplasma gallisepticum MS vlhA gene is SEQ ID NO.3-4, a probe is MS-Taq, the invention further provides a double qPCR detection method and application of the Mycoplasma gallisepticum and the Mycoplasma synoviae, the method has the advantages of high sensitivity, high specificity and simplicity and convenience in operation, and an effective means is provided for detecting and distinguishing the Mycoplasma gallisepticum and the Mycoplasma synoviae.

Description

Technical Field

[0001] The invention belongs to the technical field of Mycoplasma gallisepticum and Mycoplasma synoviae detection, and specifically relates to a dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae, and a detection method and application thereof. Background Art

[0002] Mycoplasma gallinarum has caused serious economic losses to the poultry industry. Among them, Mycoplasma gallisepticum (MG) and Mycoplasma synovialum (MS) have the strongest pathogenicity and are the most harmful, threatening the health of the poultry industry. MG, also known as avian septicemia or Mycoplasma gallisepticum, is most susceptible in chickens and turkeys. After infection, most sick chickens show respiratory symptoms such as sneezing, rales, coughing, conjunctivitis, tearing, and nasal exudation. A small number of them will experience increased embryonic mortality, reduced egg production, hatching rate, feed conversion rate, and body weight. MS, also known as Mycoplasma synovialum, can infect all kinds of poultry and wild birds, among which chickens and turkeys are the most seriously infected. After infection, the joints, tendon sheaths, synovial bursae and other parts of the sick chickens will have inflammatory reactions. Among them, laying hens will also have symptoms such as thinning, fragility, increased transparency of the eggshell, and decreased egg production and hatching rates.

[0003] In recent years, MG and MS have occurred in many provinces of my country, showing an increasing trend year by year, and the number of cases of mixed infection and invisible infection has gradually increased. In addition, MG and MS infections have also been found in chicken farms in Hainan, causing serious economic losses to farmers in Hainan.

[0004] The study found that regular monitoring, isolation and elimination of positive chickens, and strict biological control are currently the most effective measures for the prevention and control of MG and MS. However, for the monitoring of MG and MS, the clinic is in urgent need of accurate, rapid, and low-cost detection technology.

[0005] Although there are many existing MG and MS detection technologies at home and abroad, such as pathogen isolation and identification, serological detection and molecular biological detection, the pathogen isolation operation is complicated and time-consuming, and serological detection cannot distinguish between vaccine virus and wild virus, and cannot detect MG and MS at the same time; Real-time quantitative PCR (qPCR) technology is time-saving, highly sensitive, and highly specific, and can achieve quantitative detection of pathogen nucleic acids, but the cost is relatively high; multiplex qPCR technology can detect multiple diseases at the same time, greatly reducing the detection cost and shortening the detection time, and is an efficient detection method. In view of this, a dual qPCR primer set, probe and detection method that can be used for the simultaneous and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae in Hainan are developed, which is of great significance for the monitoring, detection and prevention and control of MG and MS in Hainan. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae and a detection method and application thereof in view of the deficiencies of the above-mentioned prior art. The method has the advantages of strong specificity, high sensitivity, simple and rapid operation, and economy.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae, including specific primers and probes for detecting Mycoplasma gallisepticum MG mgC2 gene and Mycoplasma synoviae MS vlhA gene; the specific primers for the Mycoplasma gallisepticum MG mgC2 gene are MG-F and MG-R, and the probe for the Mycoplasma gallisepticum MG mgC2 gene is MG-Taq; the specific primers for the Mycoplasma synoviae MS vlhA gene are MS-F and MS-R, and the probe for the Mycoplasma synoviae MS vlhA gene is MS-Taq;

[0008] The nucleotide sequence of the MG-F is shown in SEQ ID NO.1, the nucleotide sequence of the MG-R is shown in SEQ ID NO.2, the nucleotide sequence of the MS-F is shown in SEQ ID NO.3, and the nucleotide sequence of the MS-R is shown in SEQ ID NO.4;

[0009] The 5'→3' sequence of the MG-Taq is:

[0010] ROX-ATGGTTGAATCCCTTGGAATAATCG-BHQ2;

[0011] The 5'→3' sequence of the MS-Taq is:

[0012] HEX-AGACAAGTTGAAGCAACTACAACAG-BHQ1.

[0013] The present invention also provides a dual qPCR method for detecting Mycoplasma gallisepticum and Mycoplasma synoviae using the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae, the method comprising:

[0014] S1, extracting DNA of the sample to be tested;

[0015] S2, using the sample DNA to be tested described in S1 as a template, qPCR amplification was performed using MG-F, MG-R, MS-F, MS-R, MG-Taq and MS-Taq;

[0016] The reaction system of the qPCR amplification is: Premix EX Taq (Probe qPCR) 12.5 μL, sample DNA to be tested 1 μL, MG-F 0.6 μL with a concentration of 10 μmol / L, MG-R 0.6 μL with a concentration of 10 μmol / L, MS-F 0.8 μL with a concentration of 10 μmol / L, MS-R 0.8 μL with a concentration of 10 μmol / L, MG-Taq 1 μL with a concentration of 10 μmol / L, MS-Taq 0.6 μL with a concentration of 10 μmol / L, and ddH2O is added to 25 μL;

[0017] The reaction procedure of the qPCR amplification is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s; annealing at 47.8°C for 30s (collecting fluorescence); and 40 cycles;

[0018] S3, the Ct value corresponding to the drawn amplification curve is used to determine the result;

[0019] When the Ct value obtained after the amplification of the Mycoplasma gallisepticum mgC2 gene is ≤38, it means that the sample to be tested contains Mycoplasma gallisepticum; when the Ct value is >38, it means that the sample to be tested does not contain Mycoplasma gallisepticum;

[0020] When the Ct value obtained after amplifying the vlhA gene of the Mycoplasma gallinarum is ≤38, it indicates that the sample to be tested contains the Mycoplasma gallinarum, and when the Ct value is >38, it indicates that the sample to be tested does not contain the Mycoplasma gallinarum.

[0021] The present invention also provides an application of the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum, characterized in that the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum is used for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallinaceum.

[0022] The present invention also provides an application of the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum, wherein the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum is used to prepare a kit for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma gallinaceum.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a dual qPCR primer set, probe, detection method and application that can simultaneously and specifically detect Mycoplasma gallisepticum and Mycoplasma synoviae in Hainan. The method can be used to simultaneously detect Mycoplasma gallisepticum and Mycoplasma synoviae in Hainan, with strong specificity, high sensitivity, simple operation, reliable results, and can quickly distinguish between single infection and mixed infection. It can provide an effective diagnostic means for the monitoring and detection of Mycoplasma gallisepticum and Mycoplasma synoviae in Hainan, and provide reliable information for the epidemiological investigation and prevention and control of Mycoplasma gallisepticum and Mycoplasma synoviae in Hainan.

[0025] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the positive plasmid amplification results of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 2 of the present invention; wherein A is the positive plasmid amplification result of Mycoplasma gallisepticum, and B is the positive plasmid amplification result of Mycoplasma synoviae.

[0027] Figure 2 This is the single qPCR amplification result of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 3 of the present invention.

[0028] Figure 3 This is the standard curve for single qPCR amplification of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 4 of the present invention.

[0029] Figure 4 This is the single qPCR sensitivity verification result of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 5 of the present invention. Figure 4 The template amounts of A, 1-10 were 5.5×10 9 copies / μL, 5.5×10 8 copies / μL, 5.5×10 7 copies / μL, 5.5×10 6 copies / μL, 5.5×10 5 copies / μL, 5.5×10 4 copies / μL, 5.5×10 3 copies / μL, 5.5×10 2 copies / μL, 5.5×10 1copies / μL, 5.5×10 0 The plasmid with a concentration of 10 copies / μL was used as the template. Figure 4 B, the template amounts of 1-10 were 6.6×10 9 copies / μL, 6.6×10 8 copies / μL, 6.6×10 7 copies / μL, 6.6×10 6 copies / μL, 6.6×10 5 copies / μL, 6.6×10 4 copies / μL, 6.6×10 3 copies / μL, 6.6×10 2 copies / μL, 6.6×10 1 copies / μL, 6.6×10 0 copies / μL.

[0030] Figure 5 This is the standard curve for dual qPCR amplification of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 7 of the present invention.

[0031] Figure 6 The results of the dual qPCR sensitivity verification of Mycoplasma gallisepticum and Mycoplasma synoviae in Example 8 of the present invention are shown in Table 1. The template amounts of 1-9 in the figure are 4.83×10 9 copies / μL, 4.83×10 8 copies / μL, 4.83×10 7 copies / μL, 4.83×10 6 copies / μL, 4.83×10 5 copies / μL, 4.83×10 4 copies / μL, 4.83×10 3 copies / μL, 4.83×10 2 copies / μL, 4.83×10 1 copies / μL.

[0032] Figure 7 This is the specificity verification result of the dual qPCR detection method for Mycoplasma gallisepticum and Mycoplasma synoviae in Example 9 of the present invention. DETAILED DESCRIPTION

[0033] Example 1

[0034] This example is the design and synthesis of dual qPCR primers and probes for Mycoplasma gallisepticum and Mycoplasma synoviae.

[0035] Referring to multiple sequences of MG mgC2 gene and MS vlhA gene registered in GenBank, MegAlign software was used for comparative analysis, and one of the conserved sequences (MG mgC2 gene sequence accession number: KY362212.1; MS vlhA gene sequence accession number: OP850570.1) was selected and Oligo 7 software was used to design primers and probes (see Table 1). After the design was completed, it was sent to Shenggong Biotechnology (Shanghai) Co., Ltd. for synthesis. In Table 1, ROX at 5' in MG-Taq is a reporter fluorescent group linker, BHQ2 at 3' in MG-Taq is a quencher fluorescent group linker, HEX at 5' in MS-Taq is a reporter fluorescent group linker, and BHQ1 at 3' in MS-Taq is a quencher fluorescent group linker.

[0036] Table 1 Nucleotide sequences of primers and probes

[0037]

[0038] Example 2

[0039] This example is the construction of a positive plasmid.

[0040] The corresponding fragments were amplified using primers for amplifying Mycoplasma gallisepticum and Mycoplasma synoviae. The fragments amplified by MG and MS were cloned into the pMD-18T vector. After transformation, plasmid extraction and PCR identification, positive plasmids were screened out (see Figure 1 ), and the copy number of the plasmid was calculated after the concentration was determined. The initial plasmid concentration of MG was determined to be 170 ng / μL, which was converted into a copy number of 5.5×10 10 copies / μL; the initial plasmid concentration of MS was 206 ng / μL, which was converted into a copy number of 6.6×10 10 copies / μL.

[0041] Example 3

[0042] This example is the optimization of single qPCR amplification reaction conditions and reaction system.

[0043] The single primers and probes of Mycoplasma gallisepticum and Mycoplasma synoviae were evenly mixed to establish a 25 μL reaction system, and then the annealing temperature, primer concentration and probe concentration were optimized and screened.

[0044] The optimized annealing temperature gradient of MG was 50.0℃, 50.5℃, 51.4℃, 52.8℃, 54.5℃, 55.8℃, 56.6℃, and 57.0℃, respectively; the optimized primer concentration gradient was set to 0.16μmol / L, 0.20μmol / L, 0.24μmol / L, 0.32μmol / L, 0.40μmol / L, and 0.48μmol / L; the optimized probe concentration gradient was set to 0.16μmol / L, 0.20μmol / L, 0.24μmol / L, 0.32μmol / L, 0.40μmol / L, and 0.48μmol / L.

[0045] The optimized annealing temperature gradient of MS was 53.0℃, 53.5℃, 54.4℃, 55.8℃, 57.5℃, 58.8℃, 59.6℃, and 60.0℃, respectively; the optimized primer concentration gradient was set to 0.08μmol / L, 0.16μmol / L, 0.24μmol / L, 0.32μmol / L, 0.40μmol / L, and 0.48μmol / L; the optimized probe concentration gradient was set to 0.16μmol / L, 0.24μmol / L, 0.32μmol / L, 0.40μmol / L, and 0.48μmol / L.

[0046] The single qPCR detection method for Mycoplasma gallisepticum and Mycoplasma synoviae was finally determined by optimization screening (see Figure 2 The optimal primer concentrations for the PCR products are shown in Table 2.

[0047] Table 2 Optimal final concentration of each primer for single qPCR detection method

[0048]

[0049] The optimal probe concentrations are shown in Table 3.

[0050] Table 3 Optimal final concentration of each probe for single qPCR detection method

[0051] pathogen Probe concentration Mycoplasma gallisepticum MG-Taq 1.2μmol / L Mycoplasma synoviae MS-Taq 1μmol / L

[0052] The optimal reaction system of MG is shown in Table 4.

[0053] Table 4 MG reaction system

[0054] Reagents Volume (μL) MG-F (10 μmol / L) 0.8 MG-R (10 μmol / L) 0.8 MG-Taq (10 μmol / L) 1.2 DNA template 1 PremixEXTaq(ProbeqPCR) 12.5 Double distilled water Make up to 25 μL

[0055] The optimal MS reaction system is shown in Table 5.

[0056] Table 5 MS reaction system

[0057] Reagents Volume (μL) MS-F (10 μmol / L) 1.2 MS-R (10 μmol / L) 1.2 MS-Taq (10 μmol / L) 1 DNA template 1 PremixEXTaq(ProbeqPCR) 12.5 Double distilled water Fill up to 25

[0058] The optimal reaction conditions for MG were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s; annealing at 52.8°C for 30 s (collecting fluorescence); and 40 cycles.

[0059] The optimal reaction conditions for MS were: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s; annealing at 53.5°C for 30 s (collecting fluorescence); and 40 cycles.

[0060] Example 4

[0061] This example is about the establishment of a single qPCR standard curve.

[0062] After ten-fold serial dilution of the positive plasmids of MG and MS, 5.5×10 8 copies / μL, 5.5×10 7 copies / μL, 5.5×10 6 copies / μL, 5.5×10 5 copies / μL, 5.5×10 4 copies / μL, 5.5×10 3 copies / μL, 5.5×10 2 copies / μL, 5.5×10 1 copies / μL, 5.5×10 0 The concentration of template copies / μL was 6.6×10 8 copies / μL, 6.6×10 7 copies / μL, 6.6×10 6 copies / μL, 6.6×10 5 copies / μL, 6.6×10 4 copies / μL, 6.6×10 3 copies / μL, 6.6×10 2 copies / μL, 6.6×10 1 copies / μL, 6.6×10 0 The concentration of copies / μL was used as the template, and amplification was performed according to the optimized reaction conditions. The standard curve was drawn based on the amplification results (see Figure 3 ). The amplification efficiency (E) of MG was 98.3%, and the correlation coefficient (R 2 ) was 0.998, the slope was -3.364; the amplification efficiency (E) of MS was 105.9%, and the correlation coefficient (R 2 ) is 1.000 and the slope is -3.189.

[0063] Example 5

[0064] This implementation is a single qPCR sensitivity test.

[0065] MG was 5.5×10 9copies / μL, 5.5×10 8 copies / μL, 5.5×10 7 copies / μL, 5.5×10 6 copies / μL, 5.5×10 5 copies / μL, 5.5×10 4 copies / μL, 5.5×10 3 copies / μL, 5.5×10 2 copies / μL, 5.5×10 1 copies / μL, 5.5×10 0 The plasmids with the concentration of copies / μL were used as templates, and the MS was performed at 6.6×10 9 copies / μL, 6.6×10 8 copies / μL, 6.6×10 7 copies / μL, 6.6×10 6 copies / μL, 6.6×10 5 copies / μL, 6.6×10 4 copies / μL, 6.6×10 3 copies / μL, 6.6×10 2 copies / μL, 6.6×10 1 copies / μL, 6.6×10 0 The sensitivity of the established method was determined by using a plasmid with a concentration of 100 copies / μL as a template and amplifying it according to the optimized reaction conditions. Figure 4 As shown in Figure 2, the minimum detection limit of the MG qPCR assay was 5.5×10 0 copies / μL; the minimum detection limit of the MS qPCR detection method is 6.6×10 0 copies / μL.

[0066] Example 6

[0067] This example is about the optimization of primer and probe concentrations for duplex qPCR amplification reaction.

[0068] On the basis of the optimization of the single qPCR detection method, the primers and probes of Mycoplasma gallisepticum and Mycoplasma synoviae were all added to the same reaction system and mixed, and then the annealing temperature and the concentration of primers and probes were optimized and screened. The optimization gradient of annealing temperature was set to 46.0℃, 46.6℃, 47.8℃, 49.5℃, 51.7℃, 53.5℃, 54.5℃, 55.0℃, respectively; the optimization gradient of primer concentration was set to 0.16μmol / L, 0.24μmol / L, 0.32μmol / L, 0.40μmol / L and 0.48μmol / L, respectively; the optimization gradient of probe concentration was set to 0.08μmol / L, 0.16μmol / L, 0.24μmol / L, 0.32μmol / L and 0.40μmol / L, respectively. The optimal primer and probe concentrations for the dual qPCR detection method of Mycoplasma gallisepticum and Mycoplasma synoviae were finally determined through optimization screening and are shown in Table 6.

[0069] Table 6 Optimal final concentrations of primers and probes for dual qPCR detection method

[0070]

[0071] The optimal reaction system for the dual qPCR detection method is shown in Table 7.

[0072] Table 7 Reaction system of dual qPCR detection method

[0073] Reagents Volume (μL) MG-F (10 μmol / L) 0.6 MG-R (10 μmol / L) 0.6 MG-Taq (10 μmol / L) 1 MS-F (10 μmol / L) 0.8 MS-R (10 μmol / L) 0.8 MG-Taq (10 μmol / L) 0.6 DNA template 1 PremixEXTaq(ProbeqPCR) 12.5 Double distilled water Fill up to 25

[0074] The reaction conditions of the dual qPCR detection method were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s; annealing at 47.8°C for 30 s (collecting fluorescence); and 40 cycles.

[0075] Example 7

[0076] This example is the establishment of dual qPCR standard curves for Mycoplasma gallisepticum and Mycoplasma synoviae.

[0077] The positive plasmids of MG and MS were uniformly diluted to 150 ng / μL (4.83×10 10 After that, the mixture was mixed evenly in equal volumes and diluted tenfold, and 4.83×10 7 copies / μL, 4.83×10 6 copies / μL, 4.83×10 5 copies / μL, 4.83×10 4 copies / μL, 4.83×10 3 copies / μL, 4.83×10 2 copies / μL, 4.83×10 1The plasmid with a concentration of 100 copies / μL was used as a template and amplified according to the optimized reaction conditions. A standard curve was drawn based on the amplification results (see Figure 5 ). Among them, the amplification efficiency (E) and correlation coefficient (R 2 ) and slope were 109.7%, 0.998 and -3.110 respectively; the amplification efficiency (E) and correlation coefficient (R 2 ) and slope are 100%, 0.998, and -3.322, respectively.

[0078] Example 8

[0079] This example is a sensitivity test of the dual qPCR detection method.

[0080] The constructed MG and MS positive plasmids were uniformly diluted to 150 ng / μL (4.83×10 10 After that, the mixture was mixed evenly in equal volumes and diluted tenfold, and 4.83×10 9 copies / μL, 4.83×10 8 copies / μL, 4.83×10 7 copies / μL, 4.83×10 6 copies / μL, 4.83×10 5 copies / μL, 4.83×10 4 copies / μL, 4.83×10 3 copies / μL, 4.83×10 2 copies / μL, 4.83×10 1 The sensitivity of the established method was determined by using a plasmid with a concentration of 100 copies / μL as a template and amplifying it according to the optimized reaction conditions. Figure 6 As shown in Figure 2, the minimum detection limit of the MG and MS dual qPCR detection method was 4.83×10 1 copies / μL.

[0081] Example 9

[0082] This example is a specific detection of the dual qPCR detection method.

[0083] The plasmids of MG and MS were used as positive controls, and sterile distilled water was used as negative control. The detection method constructed in this application was used to perform qPCR amplification on DNA / cDNA samples of MG, MS, Newcastle disease (ND), Marek's disease (MD), infectious bursal disease (IBD), infectious laryngotracheitis (ILT) and infectious bronchitis (IBV). The results showed that only the samples of MG and MS showed positive amplification curves (see Figure 7), while other samples did not show amplification curves, indicating that the detection method of the present invention has no cross-reaction with other viruses and has strong specificity.

[0084] Example 10

[0085] Reproducibility testing of duplex qPCR assay

[0086] The concentration was selected as 10 8 copies / μL, 10 6 copies / μL and 10 4 The plasmid with 100 copies / μL was used as a template, and amplified by the dual qPCR detection method constructed in this application, and repeated three times to verify the intra-batch repeatability of this method; then the three templates were amplified at different times to verify the inter-batch repeatability. The repeatability of this method was evaluated by calculating the mean, standard deviation and coefficient of variation of the Ct value of the amplification result. The results show (see Table 8) that the intra-batch repeatability and inter-batch repeatability coefficients of MG and MS are both below 1.50%, indicating that the established method has good repeatability.

[0087] Table 8 MG, MS dual qPCR repeatability experimental results

[0088]

[0089] Embodiment 11

[0090] This example is the application and accuracy verification of the dual qPCR detection method.

[0091] The method of the present invention was used to detect 96 clinical samples (including throat swabs, tracheal swabs, and lung tissues) collected from a chicken farm in Wenchang, Hainan Province, and 13 positive samples of Mycoplasma gallisepticum and 31 positive samples of Mycoplasma synoviae were detected. The test results were compared with the test results of the MS qPCR detection method and the MG qPCR detection kit published by the agricultural industry standard of the People's Republic of China (see Table 9). The detection result conformity rate of MG in the two detection methods reached 100%, and the detection result conformity rate of MS reached 95.74%. The positive detection number of the established method was higher than that of the national standard method, indicating that the established method has high accuracy and can be used for clinical detection.

[0092] Table 9 Comparison of the detection results of the dual qPCR detection method and the single qPCR detection method

[0093]

[0094]

[0095] The dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum provided by the present invention can be used for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallinaceum, and can be used for preparing a kit for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallinaceum.

[0096] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A dual qPCR primer combination for Mycoplasma gallisepticum and Mycoplasma synoviae, characterized in that: The invention comprises specific primers and probes for detecting the Mycoplasma gallisepticum MG mgC2 gene and the Mycoplasma synoviae MS vlhA gene; the specific primers for the Mycoplasma gallisepticum MG mgC2 gene are MG-F and MG-R, and the probe for the Mycoplasma gallisepticum MG mgC2 gene is MG-Taq; the specific primers for the Mycoplasma synoviae MS vlhA gene are MS-F and MS-R, and the probe for the Mycoplasma synoviae MS vlhA gene is MS-Taq; The nucleotide sequence of the MG-F is shown in SEQ ID NO.1, the nucleotide sequence of the MG-R is shown in SEQ ID NO.2, the nucleotide sequence of the MS-F is shown in SEQ ID NO.3, and the nucleotide sequence of the MS-R is shown in SEQ ID NO.4; The 5'→3' sequence of the MG-Taq is: ROX-ATGGTTGAATCCCTTGGAATAATCG-BHQ2; The 5'→3' sequence of the MS-Taq is: HEX-AGACAAGTTGAAGCAACTACAACAG-BHQ1.

2. A dual qPCR method for detecting Mycoplasma gallisepticum and Mycoplasma synoviae using the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae according to claim 1, characterized in that: The method is: S1, extracting DNA of the sample to be tested; S2, using the sample DNA to be tested described in S1 as a template, qPCR amplification was performed using MG-F, MG-R, MS-F, MS-R, MG-Taq and MS-Taq; The reaction system of the qPCR amplification is: Premix EX Taq 12.5 μL, sample DNA 1 μL, MG-F 0.6 μL at a concentration of 10 μmol / L, MG-R 0.6 μL at a concentration of 10 μmol / L, MS-F 0.8 μL at a concentration of 10 μmol / L, MS-R 0.8 μL at a concentration of 10 μmol / L, MG-Taq 1 μL at a concentration of 10 μmol / L, MS-Taq 0.6 μL at a concentration of 10 μmol / L, and ddH2O is added to 25 μL. The reaction procedure of the qPCR amplification is: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s; annealing at 47.8°C for 30s, while collecting fluorescence; 40 cycles; S3, the Ct value corresponding to the drawn amplification curve is used to determine the result; When the Ct value obtained after the amplification of the Mycoplasma gallisepticum mgC2 gene is ≤38, it means that the sample to be tested contains Mycoplasma gallisepticum; when the Ct value is >38, it means that the sample to be tested does not contain Mycoplasma gallisepticum; When the Ct value obtained after amplifying the vlhA gene of the Mycoplasma gallinarum is ≤38, it indicates that the sample to be tested contains the Mycoplasma gallinarum, and when the Ct value is >38, it indicates that the sample to be tested does not contain the Mycoplasma gallinarum.

3. A use of the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae as claimed in claim 1, characterized in that: The dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma gallinaceum is used for simultaneous detection of Mycoplasma gallisepticum and Mycoplasma gallinaceum.

4. A use of the dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae as claimed in claim 1, characterized in that: The dual qPCR primer combination of Mycoplasma gallisepticum and Mycoplasma synoviae is used to prepare a kit for simultaneously detecting Mycoplasma gallisepticum and Mycoplasma synoviae.

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