Primer probe combination for detecting avian mycoplasma, kit, detection method and application

Abstract

The invention discloses a primer probe combination for detecting avian mycoplasma, the avian mycoplasma comprises one or more of an MG attenuated vaccine strain F-36, an MG clinical strain, an MS attenuated vaccine strain MSH or an MS clinical strain, and the sequence of the primer probe is shown as SEQ ID NO.1-SEQ ID NO.8. The invention further discloses a kit for detecting the avian mycoplasma. The primer probe combination can be used for rapidly and accurately detecting an MG attenuated vaccine strain F-36, an MG clinical strain, an MS attenuated vaccine strain MSH and an MS clinical strain, and is high in specificity, high in sensitivity and good in repeatability. In addition, only about one hour is needed for completing one-time sample detection and result interpretation, and the detection cost can be controlled to be 1-3 yuan / sample.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and particularly relates to a primer-probe combination, a kit, a detection method and an application for detecting avian mycoplasma. Background Art

[0002] Avian mycoplasma is a pathogen that seriously endangers the health of poultry. Among them, Mycoplasma gallisepticum (MG) infection mainly causes chronic respiratory disease (CRD), and the clinical manifestations are cough, runny nose, swelling of the infraorbital sinus, dyspnea, slow growth of young chickens and reduced feed conversion rate. Mycoplasma synoviae (MS) infection mainly causes infectious synovitis and subclinical respiratory infection, and the typical symptoms are joint swelling (tarsal joint, claw pad), lameness, and sternal cyst. After avian mycoplasma infection, the egg production rate of breeding chickens will decrease, the eggshell quality will deteriorate, and the hatching rate will decrease, and it can spread through vertical transmission (breeding eggs) and horizontal transmission (aerosol, contact). At present, the prevention and control of breeding chickens mainly rely on live vaccine immunization, and the MG attenuated vaccine strain F-36 and the MS attenuated vaccine strain MSH play an important role in the prevention and control of avian mycoplasma.

[0003] At present, multiplex fluorescence quantitative PCR technology has become one of the main means for detecting pathogens and evaluating vaccine efficacy. Since the isolation of MG and MS requires complex culture media (such as Frey's medium), has slow growth (5-21 days), and requires a CO 2 environment. Although the plate agglutination test (SPA) or ELISA is rapid, it cannot distinguish current infection from vaccine antibodies and is insensitive to early infection (antibodies not yet produced). Single PCR requires multiple reactions to identify pathogens, which is time-consuming and prone to contamination, lacks quantitative ability, and is difficult to evaluate the degree of infection. Although a dual fluorescence quantitative PCR method for the MS vaccine strain MSH and the MS clinical strain has been developed, the existing methods cannot meet the needs of farms for rapid identification of MG, MS and mixed infections, especially lack a single-tube synchronous quantitative detection scheme. The present invention designs a multiplex probe-based fluorescence quantitative PCR to break through the technical bottleneck and provide a key tool for the precise prevention and control of avian mycoplasma infection. Summary of the Invention

[0004] The purpose of the present invention is to provide a primer-probe combination, a kit, a detection method and an application for detecting avian mycoplasma, so as to meet the needs of rapid identification of MG, MS and mixed infections, especially the purpose of single-tube synchronous quantitative detection.

[0005] According to the first aspect of the present invention, a primer-probe combination for detecting avian mycoplasma is provided. The avian mycoplasma includes one or more of the MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH, or MS clinical strain. The sequences of the primers and probes are shown in SEQ ID NO.1-SEQ ID NO.8. Thus, through this primer-probe combination, single-tube synchronous quantitative detection can be achieved by multiplex fluorescence quantitative PCR detection technology. It can quickly identify the situations of MG, MS, and mixed infections. Moreover, combined with the antibody detection situation, it can distinguish between current infections and antibodies produced by vaccines (it can effectively distinguish between vaccine strains and clinical strains), the detection is more accurate, it can better grasp the current clinical epidemic pattern, strengthen the prevention and control of avian mycoplasma, reduce the incidence rate, and reduce economic losses.

[0006] In some embodiments, the primer sequences for MG are shown in SEQ ID NO.1 and SEQ ID NO.2, the probe sequence for the MG attenuated vaccine strain F-36 is shown in SEQ ID NO.3, and the probe sequence for the MG clinical strain is shown in SEQ ID NO.4; the primer sequences for MS are shown in SEQ ID NO.5 and SEQ ID NO.6, the probe sequence for the MS attenuated vaccine strain MSH is shown in SEQ ID NO.7, and the probe sequence for the MS clinical strain is shown in SEQ ID NO.8.

[0007] In some embodiments, the probe fluorescence reporter group for the MG attenuated vaccine strain F-36 is CY5, the probe fluorescence reporter group for the MG clinical strain is FAM, the probe fluorescence reporter group for the MS attenuated vaccine strain MSH is ROX, and the probe fluorescence reporter group for the MS clinical strain is VIC.

[0008] According to the second aspect of the present invention, a kit for simultaneously detecting different types of avian mycoplasma is provided. The kit includes the above-mentioned primer-probe combination. Through this kit, the MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH, and MS clinical strain can be detected quickly and accurately, and it has strong specificity, high sensitivity, and good repeatability. Moreover, single-tube synchronous quantitative detection can be achieved, it can quickly identify the situations of MG, MS, and mixed infections. Moreover, combined with the antibody detection situation, it can distinguish between current infections and antibodies produced by vaccines (it can effectively distinguish between vaccine strains and clinical strains), the detection is more accurate, it can better grasp the current clinical epidemic pattern, strengthen the prevention and control of avian mycoplasma, reduce the incidence rate, and reduce economic losses.

[0009] In some embodiments, the kit further includes other reagents for realizing multiplex fluorescence quantitative PCR detection.

[0010] According to the third aspect of the present invention, there is provided an application of the primer-probe combination or kit in simultaneously detecting different species of avian mycoplasmas, wherein the different species of avian mycoplasmas include one or more of the MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain, and the application is not for the purpose of disease diagnosis or treatment. By this application, four different species of avian mycoplasmas can be rapidly and accurately detected, the infection of pathogens can be timely discovered and diagnosed, which helps to take corresponding prevention and control measures and effectively reduce the impact brought by avian mycoplasmas.

[0011] According to the fourth aspect of the present invention, there is provided an application of the primer-probe combination in preparing a product for detecting the infection of one or more mycoplasmas among the MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain. By this application, a product that can be used to detect the infection of one or more mycoplasmas among the MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain can be prepared. The use of this product can improve the detection efficiency of farms, rapidly and accurately detect four different species of avian mycoplasmas, timely discover and diagnose the infection of pathogens, which helps to take corresponding prevention and control measures and effectively reduce the impact brought by avian mycoplasmas.

[0012] According to the fifth aspect of the present invention, there is provided a method for simultaneously detecting different species of avian mycoplasmas, and the method includes the following steps:

[0013] S1: Synthesize the primer-probe combination or use the kit;

[0014] S2: Establish a multiplex fluorescence quantitative PCR system: the final concentration of the primer in the reaction system of multiplex fluorescence quantitative PCR detection is 0.4 μmol / L, and the final concentration of the probe is 0.2 μmol / L;

[0015] S3: The reaction program of multiplex fluorescence quantitative PCR detection is: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 sec, annealing and extension at 62.2 °C for 30 sec, 40 cycles, and the fluorescence signal is collected after each extension ends;

[0016] S4: The result determination method of multiplex fluorescence quantitative PCR detection is: when the Cq value of the detected sample ≤ 37 and the amplification curve shows an obvious exponential growth phase, the measurement result is valid and the detected sample is determined to be positive; when the Cq value of the detected sample > 37 or the Cq value of the sample cannot be detected, the detected sample is determined to be negative.

[0017] Thus, by this method, the detection efficiency and accuracy of avian mycoplasma can be improved, and the immune effect of chicken flocks immunized with F-36 strain or MSH strain can be tracked. It has strong specificity and high sensitivity. It can better grasp the current clinical epidemic law, strengthen the prevention and control of avian mycoplasma, reduce the incidence rate, and reduce economic losses.

[0018] In some embodiments, the positive control plasmid for the multiplex fluorescence quantitative PCR detection is the pMD18T recombinant plasmid, and the pMD18T recombinant plasmid is constructed by inserting the sequence shown in SEQ ID NO.9 into the pMD18T vector.

[0019] In some embodiments, the multiplex fluorescence quantitative PCR is performed by detecting whether the target gene of at least one of the attenuated vaccine strain F-36 of Mycoplasma gallisepticum, the clinical strain of MG, the attenuated vaccine strain MSH of Mycoplasma synoviae or the clinical strain of MS is contained in the sample.

[0020] Advantages of the present invention:

[0021] 1. The primer-probe combination, kit or detection method disclosed in the present invention can simultaneously detect 4 different avian mycoplasmas. In terms of the detection itself, it is much more difficult to identify 4 types of avian mycoplasmas in a single system than to detect 1 or 2 types. Moreover, since MS and MG belong to the same genus but different species and are very similar at the gene level, it is very difficult to screen out such non-crossing target points, and it is even more difficult to screen out primers and probes for efficient detection. In addition, in the present invention, the vaccine strain and the clinical strain can be effectively detected, which is equivalent to distinguishing the same species and origin, and can only be achieved by screening point mutations. In the present invention, through a bioinformatics method, more than 10,000 point mutation target points of MS and MG are constructed, and then the feasibility of the primer-probe sequences is analyzed by software, and combined with the point mutation conditions of the vaccine strain and the clinical strain, and finally the best primer and probe sequence combination is screened out.

[0022] 2. The design of the probe in the present invention is particularly important. The probe needs to cooperate with the primer. The probe needs to bind to the template prior to the primer, otherwise it will cause the DNA polymerase to be unable to cleave the probe and unable to generate a fluorescence signal, resulting in detection failure. At the same time, the probe needs to ensure efficient binding to the template, otherwise it cannot effectively bind to the template, also resulting in the inability to generate a fluorescence signal and detection failure; at the same time, it is necessary to ensure that there is no polymerization and mismatch between the probes and between the probe and the primer.

[0023] 3. The primer-probe combination, kit, or detection method disclosed in the present invention can achieve single-tube synchronous quantitative detection, can quickly identify the situations of MG, MS, and mixed infections, and can also distinguish between current infections and antibodies produced by vaccines (it can effectively distinguish between vaccine strains and clinical strains). The detection is more accurate, can better grasp the current clinical epidemic pattern, strengthen the prevention and control of avian mycoplasma, reduce the incidence rate, and mitigate economic losses.

[0024] 4. The primer-probe combination, kit, or detection method disclosed in the present invention can quickly and accurately detect four different types of avian mycoplasma, timely discover and diagnose pathogen infections, help take corresponding prevention and control measures, and effectively reduce the impact brought by avian mycoplasma.

[0025] 5. The primer-probe combination, kit, or detection method disclosed in the present invention can improve the detection efficiency and accuracy of avian mycoplasma, and track the immune effects of chicken flocks immunized with the F-36 strain or MSH strain. It has strong specificity and high sensitivity. It can better grasp the current clinical epidemic pattern, strengthen the prevention and control of avian mycoplasma, reduce the incidence rate, and mitigate economic losses.

[0026] 6. For the primer-probe combination, kit, or detection method disclosed in the present invention, its detection specificity can reach 100%, and the sensitivities can reach 8.64 copies / μL, 9.26 copies / μL, 6.67 copies / μL, and 7.11 copies / μL respectively. And it only takes about 1 hour to complete the detection of a sample and the result interpretation, and the detection cost can be controlled within 1 - 3 yuan per sample. Description of the Drawings

[0027] Figure 1 It is the detection result graph when screening the primer-probe combination in Example 1 of the present invention;

[0028] Figure 2 It is the specific experiment result graph of the primer-probes of four different types of avian mycoplasma in Example 2 of the present invention: Among them, line A is the amplification curve of the MG attenuated vaccine strain F-36 labeled with CY5 fluorescence, line B is the amplification curve of the MG clinical strain labeled with FAM fluorescence, line C is the amplification curve of the MS attenuated vaccine strain MSH labeled with ROX fluorescence, and line D is the amplification curve of the MS clinical strain labeled with VIC fluorescence;

[0029] Figure 3 It is the standard curve graph of the positive control plasmid of the primer-probes of four different types of avian mycoplasma in Example 3 of the present invention: Among them, Figure 3 A is the standard curve of the MG attenuated vaccine strain F-36 labeled with CY5 fluorescence, Figure 3 B is the standard curve of the MG clinical strain labeled with FAM fluorescence, Figure 3C is the standard curve of fluorescence labeling of MS attenuated vaccine strain MSH ROX, Figure 3 D is the standard curve of VIC fluorescence labeling of MS clinical strain;

[0030] Figure 4 The results of the intra-batch and inter-batch repeatability test of four different types of avian mycoplasma primer probes in Example 4 of the present invention are shown in FIG. Figure 4 A is the fluorescence labeling standard curve of MG attenuated vaccine strain F-36CY5, Figure 4 B is the FAM fluorescence labeling standard curve of MG clinical strain, Figure 4 C is the standard curve of fluorescence labeling of MS attenuated vaccine strain MSH ROX, Figure 4 D is the standard curve of VIC fluorescence labeling of MS clinical strains. DETAILED DESCRIPTION

[0031] The invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] Example 1. Establishment of a multiplex fluorescence quantitative PCR detection method for simultaneous detection of four different types of avian mycoplasma

[0033] 1.1 Genome preparation

[0034] Four different types of avian mycoplasma DNA were extracted respectively, and all DNAs were stored at -80°C for future use. The four different types of avian mycoplasmas were Mycoplasma gallisepticum (MG) attenuated vaccine strain F-36, MG clinical strain, Mycoplasma synoviae (MS) attenuated vaccine strain MSH, and MS clinical strain; among them, MG attenuated live vaccine F-36 was purchased from Zhaofenghua Biotechnology Co., Ltd., product name: Hubangning, batch number: 22121001; MS attenuated live vaccine MSH was purchased from Shandong Xinde Technology Co., Ltd., product name: MSH live vaccine, batch number: MSH213171AG; MG clinical strain was isolated from tracheal samples of incomplete live embryos of chickens that appeared in hatcheries in Guangdong Province; MS clinical strain was isolated from arthritis samples of chickens collected in Guangdong Province.

[0035] The steps for the isolation and identification of MG and MS clinical strains are as follows: the collected samples are added to the mycoplasma liquid culture medium for grinding or sufficient shaking, filtered with a 0.45μm bacterial filter and added with an appropriate amount of mycoplasma liquid culture medium and placed in a 37℃ constant temperature incubator for culture. Continue to observe until the color of the liquid culture medium changes from red to orange-yellow, then take 20μL and drop it into the mycoplasma solid culture medium and place it in a 37℃ constant temperature incubator for culture for 5-7 days. The strain with a fried egg-like colony observed under a 200X microscope is successfully isolated. Pick a single colony for culture enrichment, and identify the strain by fluorescent quantitative PCR.

[0036] 1.2 Primer and probe design and synthesis:

[0037] The published gene sequences of four avian mycoplasmas, namely the MG attenuated vaccine strain F-36, the MG clinical strain, the MS attenuated vaccine strain MSH, and the MS clinical strain, were respectively compared in GenBank (the GenBank gene numbers of different types of strains are shown in Table 1). The pan-genome analysis of Mycoplasma gallisepticum and Mycoplasma synoviae was carried out using roary v.3.13.0 to determine the differential gene fragments between the two species. The seqkit v2.8.0 software was used to extract the differential gene fragments. Snippy v4.6.0 was used to compare the mutation sites between the MG attenuated vaccine strain F-36 and the MG clinical strain, and approximately 17,000 SNP sites were collected in total; similarly, 19,000 SNP sites were collected for the comparison between the MS attenuated vaccine strain MSH and the MS clinical strain. Subsequently, the target sites were corresponding in the differential gene fragments, and the Primer Premier 5.0 was used to evaluate and screen the primers and probes in the specific regions. Specific primers and probes were designed. The 5' end of the probe was labeled with CY5, FAM, ROX, or VIC fluorescent emission groups, and the 3' end was labeled with BHQ1 or BHQ2 fluorescent quenching groups. A total of 18 sets of primer and probe combinations were designed. By performing qPCR amplification on the nucleic acid DNA of the standard samples, namely the MG attenuated vaccine strain F-36, the MG clinical strain, the MS attenuated vaccine strain MSH, and the MS clinical strain, it was found that non-specific amplification or no amplification occurred in 17 sets for single positive samples, as Figure 1 shown. Finally, only 1 set of the best primer and probe sequence combination without non-specific amplification, as shown in SEQ ID NO.1 - SEQ ID NO.8 (Table 2), was screened out. A pMD18T recombinant plasmid containing the target genes of the four avian mycoplasmas (MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH, MS clinical strain) was constructed, and the primer, probe, and target gene sequences were synthesized by BGI.

[0038] Table 1 GenBank gene numbers of different types of strains

[0039]

[0040] Table 2 Primer table

[0041]

[0042] The target gene sequences of the four avian mycoplasmas (MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH, MS clinical strain) are as shown in SEQ ID NO.9:

[0043] GTCCCCAAGCAAAACCGTGACGTGCTAGTTTATTCACCTGAggtcGGCGGTTTTGCTGGGGTGTTTggtaAAGTTTTTGCCTAACTCATTGCTGGGGTGCTTaatgAGTTTTTACCTAACTCAGCTGGTGATGTTAGTATTGATCGTTTTcctaAACTACTAGGGGAGCCTAATGATGTTAGAGTAATAGGAGCTATGAAAATGGTggctTGGCTGAGATGAACTGTTCAGACCCACAGGTGCTAATACCGCATAATggctTTATGACCTGATTTGGTTAGTACCACCGCATGGTAGATGGATGAAAGGCttagGTGCATTAGCTAGTTGGTGGG。

[0044] Construction of pMD18T recombinant plasmid: The above four Mycoplasma gallisepticum target gene sequences (SEQ ID NO.9) were inserted into the pMD18T vector (TaKaRa product number: D101A) to construct the pMD18T recombinant plasmid. This pMD18T recombinant plasmid contains the target genes of 4 mycoplasmas, namely the attenuated MG vaccine strain F-36, the clinical MG strain, the attenuated MS vaccine strain MSH, and the clinical MS strain. The nucleotide sequence of this pMD18T recombinant plasmid is shown in SEQ ID NO.10.

[0045] 1.3 Establishment of multiplex fluorescence quantitative PCR system:

[0046] Using the pMD18T recombinant plasmid as the positive control plasmid for multiplex fluorescence quantitative PCR reaction, the amplification reaction system was 20 μL, including 10 μL of ABclonal 2×Taq DNA Polymerase MIX enzyme, 0.4 μmol / L of each upstream and downstream primer, 0.2 μmol / L of the probe, 4 μL of template DNA, and deionized water was added to make up 20 μL; The reaction procedure was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing and extension at 62.2°C for 30 s, 40 cycles, and the fluorescence signal was collected after each extension ended. At the same time, a blank control group of deionized water was set up.

[0047] Example 2, Specificity verification of primers and probes

[0048] Using the attenuated MG vaccine strain F-36, MG clinical strain, attenuated M. synoviae vaccine strain MSH, M. synoviae clinical strain, and cDNA or DNA of other pathogens as templates, including avian susceptible pathogens such as avian Marek's disease virus, avian leukosis virus, Newcastle disease virus, adenovirus, infectious bronchitis virus of chickens, Escherichia coli, Salmonella, Escherichia coli, Salmonella, Staphylococcus, etc., each reaction system was 20 μL, including 10 μL of 2×TaqDNA Polymerase MIX enzyme, 0.4 μmol / L each of upstream and downstream primers, 0.2 μmol / L of probe, 4 μL of template DNA, and deionized water was added to make up 20 μL; the reaction procedure was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing and extension at 62.2°C for 30 s. After 40 cycles, fluorescence signal collection was performed at the end of each extension. Using the pMD18T recombinant plasmid as a positive control and sterile double-distilled water as a negative control. The experimental results showed that there were no amplification signals in the negative control and non-target pathogen samples. The experimental results are as Figure 2 shown, Figure 2 In Figure 2 , line A is the amplification curve of the attenuated MG vaccine strain F-36 labeled with CY5 fluorescence; line B is the amplification curve of the MG clinical strain labeled with FAM fluorescence; line C is the amplification curve of the attenuated M. synoviae vaccine strain MSH labeled with ROX fluorescence; line D is the amplification curve of the M. synoviae clinical strain labeled with VIC fluorescence. The results showed that the specificity of the fluorescence quantitative PCR detection in the present invention was 100%.

[0049] Example 3. Sensitivity evaluation of primers and probes

[0050] Using the pMD18T recombinant plasmid as a template, it was diluted with sterile double-distilled water according to the attenuated Mycoplasma gallisepticum vaccine strain F-36: 8.64×10 5 copies / μL - 8.64×10 1 copies / μL; MG clinical strain: 9.26×10 5 copies / μL - 9.26×10 1 copies / μL; attenuated Mycoplasma synoviae vaccine strain MSH: 6.67×10 5 copies / μL - 6.67×10 1 copies / μL; M. synoviae clinical strain: 7.11×10 5 copies / μL - 7.11×10 1 copies / μL, and then multiplex fluorescence quantitative PCR was performed for 5 gradients to determine the sensitivity of the multiplex fluorescence quantitative PCR method.

[0051] According to the relationship between the concentration of the pMD18T recombinant plasmid and the Cq value, a standard curve was made. The results are as Figure 3 shown in Table 3, Figure 3A is the standard curve of fluorescence labeling of the attenuated MG vaccine strain F-36CY5, Figure 3 B is the standard curve of fluorescence labeling of the clinical MG strain FAM, Figure 3 C is the standard curve of fluorescence labeling of the attenuated MS vaccine strain MSH ROX, Figure 3 D is the standard curve of fluorescence labeling of the clinical MS strain VIC. The results show that the sensitivities of this method can reach 8.64 copies / μL (attenuated MG vaccine strain F-36), 9.26 copies / μL (clinical MG strain), 6.67 copies / μL (attenuated MS vaccine strain MSH), and 7.11 copies / μL (clinical MS strain), respectively.

[0052] Table 3 Indexes of the standard curves of the primer-probe of each fluorescence signal

[0053]

[0054] Example 4. Evaluation of the repeatability of the primer-probe

[0055] Using the pMD18T recombinant plasmid as the template, it was diluted with sterile double-distilled water according to the attenuated MG vaccine strain F-36: 8.64×10 5 copies / μL; clinical MG strain: 9.26×10 5 copies / μL; attenuated MS vaccine strain MSH: 6.67×10 5 copies / μL; clinical MS strain: 7.11×10 5 copies / μL, and the between-batch and within-batch repeatability tests of multiplex fluorescence quantitative PCR were carried out. The results are as Figure 4 shown, Figure 3 A is the standard curve of fluorescence labeling of the attenuated MG vaccine strain F-36CY5, Figure 4 B is the standard curve of fluorescence labeling of the clinical MG strain FAM, Figure 4 C is the standard curve of fluorescence labeling of the attenuated MS vaccine strain MSH ROX, Figure 4 D is the standard curve of fluorescence labeling of the clinical MS strain VIC. The coefficients of variation between batches and within batches of the primer-probe for each item were calculated as shown in Table 4. The results show that the standard deviations between batches and within batches of this method are small, and the coefficients of variation are all less than 1%, indicating good repeatability and significant stability.

[0056] Table 4 Results of the evaluation of the within-batch and between-batch repeatability of the primer-probe of each fluorescence signal

[0057]

[0058]

[0059] Example 5. Detection of clinical samples with the primer-probe

[0060] The method established in Example 1 was used to detect clinical samples.

[0061] There were a total of 60 clinical samples: swabs from the upper respiratory tract of chickens in the Guangdong region from November 2024 to January 2025.

[0062] Nucleic acid DNA was extracted from 60 chicken upper respiratory tract swabs, and multiplex fluorescence quantitative PCR detection was performed according to the method and primer-probe combination of Example 1.

[0063] The results of multiplex fluorescence quantitative PCR detection showed that among the 60 samples, 40 were identified as the attenuated MG vaccine strain F-36, 5 were MG clinical strains, 55 were the attenuated MS vaccine strain MSH, and 0 were MS clinical strains. Moreover, this method can detect 94 samples simultaneously each time, and it only takes about 1 hour to complete the detection of one sample and the result interpretation. In addition, the detection cost of nucleic acid is about 1.5 yuan per sample. This shows that this method has high detection efficiency, can effectively distinguish the attenuated MG vaccine strain F-36, MG clinical strain, attenuated MS vaccine strain MSH, and MS clinical strain, and has a low detection cost, so it can be widely promoted and used.

[0064] As can be seen from the above examples, the specific primer-probe combination designed in the present invention for simultaneously detecting the attenuated MG vaccine strain F-36, MG clinical strain, attenuated MS vaccine strain MSH, and MS clinical strain has strong specificity, and is negative for detecting pathogens other than these four avian mycoplasmas, with a detection specificity of 100%; it has high sensitivity, and can reach 8.64 copies / μL, 9.26 copies / μL, 6.67 copies / μL, and 7.11 copies / μL respectively for the detection of these four categories; it has remarkable stability, and the coefficient of variation within and between batches is less than 1%.

[0065] The design of the probe in the present invention is particularly important. The probe needs to cooperate with the primer. The probe needs to bind to the template prior to the primer, otherwise it will cause the DNA polymerase to be unable to cleave the probe and unable to generate a fluorescence signal, resulting in detection failure. At the same time, the probe needs to ensure efficient binding to the template, otherwise it cannot effectively bind to the template, also resulting in the inability to generate a fluorescence signal and causing detection failure; at the same time, it is necessary to ensure that there is no polymerization and mismatch between the probes and between the probe and the primer.

[0066] The target gene selected for detection in the present invention is reasonable, and the design and combination of primers and probes are excellent. It can accurately, specifically and sensitively detect four different avian mycoplasmas simultaneously, providing an effective detection means for mastering the infection situation of avian mycoplasmas and the vaccine immunization efficacy. The detection has strong specificity, high sensitivity and good repeatability. It can perform quantitative detection of pathogens, which is fast, simple, time-saving and labor-saving. Moreover, it can achieve single-tube synchronous quantitative detection, quickly identify the situations of MG, MS and mixed infections, and combined with the antibody detection situation, it can distinguish whether it is a current infection or an antibody produced by the vaccine (it can effectively distinguish the vaccine strain and the clinical strain), the detection is more accurate, can better master the current clinical epidemic law, strengthen the prevention and control of avian mycoplasmas, reduce the incidence rate and reduce economic losses.

[0067]

Claims

1. A primer-probe combination for detecting avian mycoplasma, wherein: The avian mycoplasma includes one or more of MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain, and the sequence of the primer probe combination is shown in SEQ ID NO.1-SEQ ID NO.

8.

2. The primer-probe combination according to claim 1, wherein: The primer sequences for MG are shown in SEQ ID NO.1 and SEQ ID NO.2, the probe sequence of the MG attenuated vaccine strain F-36 is shown in SEQ ID NO.3, and the probe sequence of the MG clinical strain is shown in SEQ ID NO.4; the primer sequences for MS are shown in SEQ ID NO.5 and SEQ ID NO.6, the probe sequence of the MS attenuated vaccine strain MSH is shown in SEQ ID NO.7, and the probe sequence of the MS clinical strain is shown in SEQ ID NO.

8.

3. The primer-probe combination according to claim 2, wherein: The probe fluorescent reporter group for the MG attenuated vaccine strain F-36 is CY5, the probe fluorescent reporter group for the MG clinical strain is FAM, the probe fluorescent reporter group for the MS attenuated vaccine strain MSH is ROX, and the probe fluorescent reporter group for the MS clinical strain is VIC.

4. A kit for simultaneously detecting different types of avian mycoplasma, wherein: The kit comprises the primer-probe combination described in any one of claims 1 to 3.

5. The kit according to claim 4, wherein The kit also includes other reagents for realizing multiplex fluorescence quantitative PCR detection.

6. Use of the primer-probe combination described in any one of claims 1 to 3 or the kit described in claim 4 or 5 for simultaneously detecting different types of avian mycoplasma, wherein the different types of avian mycoplasma include one or more of MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain, and the use is not for the purpose of disease diagnosis or treatment.

7. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a product for detecting one or more mycoplasma infections in MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain.

8. A method for simultaneously detecting different types of avian mycoplasma, wherein: The method comprises the following steps: S1: synthesizing the primer-probe combination described in any one of claims 1 to 3, or using the kit described in claim 4 or 5; S2: Establishment of multiplex fluorescence quantitative PCR system: The final concentration of primers in the reaction system of multiplex fluorescence quantitative PCR detection is 0.4 μmol / L, and the final concentration of probes is 0.2 μmol / L; S3: The reaction procedure of multiplex fluorescence quantitative PCR detection was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing and extension at 62.2°C for 30 sec, 40 cycles, and fluorescence signal collection was performed after each extension. S4: The result of the multiplex fluorescence quantitative PCR test is determined as follows: when the Cq value of the test sample is ≤37 and the amplification curve shows an obvious exponential growth period, the test result is valid and the test sample is determined to be positive; when the Cq value of the test sample is greater than 37 or the sample Cq value cannot be detected, the test sample is determined to be negative.

9. The method according to claim 8, wherein: The positive control plasmid for the multiplex fluorescence quantitative PCR detection is a pMD18T recombinant plasmid, and the pMD18T recombinant plasmid is constructed by inserting the sequence shown in SEQ ID NO.9 into the pMD18T vector.

10. The method according to claim 8 or 9, wherein: The multiplex fluorescence quantitative PCR is performed by detecting whether the sample contains at least one target gene of MG attenuated vaccine strain F-36, MG clinical strain, MS attenuated vaccine strain MSH or MS clinical strain.

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