Real-time fluorescence quantitative PCR kit for differentiating wild-type Mycoplasma gallisepticum strains from F, TS-11, and 6 / 85 strains

Abstract

The present invention discloses a real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from the F, TS-11, and 6 / 85 strains. The kit has high sensitivity, strong specificity, and good repeatability, and can accurately distinguish wild-type Mycoplasma gallisepticum strains from vaccine strains (F, TS-11, and 6 / 85). It can be used to distinguish isolated, cultured, and purified Mycoplasma gallisepticum isolates, assess the in vivo replication efficiency of attenuated live vaccines (F, TS-11, and 6 / 85) in immunized chickens, and monitor wild-type Mycoplasma infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, relates to a fluorescent quantitative PCR kit and an application thereof, and particularly relates to a real-time fluorescent quantitative PCR kit for distinguishing wild strains of Mycoplasma gallisepticum from F strains, TS-11 strains, and 6 / 85 strains. Background Art

[0002] Mycoplasma gallisepticum (MG) is the most pathogenic and economically damaging avian mycoplasma pathogen. The economic losses to the poultry industry primarily include reduced chicken meat quality, carcass quality, egg production in laying hens, reduced feed conversion efficiency in commercial chickens, decreased eggshell quality, reduced embryo hatchability in breeder hens, disease control costs including vaccines and medications, disease surveillance costs, and the loss of culled chickens. Studies have shown that MG infection can reduce egg production in laying hens by 10%-20%, reduce broiler weight gain by 10%-20%, reduce feed conversion by 10%-20%, increase mortality by 5%-10%, and increase embryonic mortality by 5%-10%. Furthermore, MG infection can severely impact international trade. Consequently, MG has become one of the most costly infectious diseases facing the global poultry industry.

[0003] MG can be transmitted both vertically and horizontally. The primary prevention and control approach for vertically transmitted infections is stock purification. While some developed countries, such as the United States, have largely eliminated MG from their breeding flocks through poultry improvement programs, this approach is more challenging to implement in my country. Currently, the primary method for preventing and controlling MG infection in my country is vaccination, with the most widely used attenuated live vaccines, including the F, TS-11, and 6 / 85 strains.

[0004] With the introduction of attenuated live vaccines, a new challenge urgently needs to be addressed: how to distinguish wild-type and vaccine strains and evaluate vaccination efficacy. With the advancement of sequencing technology and the isolation of MG strains, whole-genome sequencing of wild-type and vaccine strains has been completed. Wild-type and vaccine strains exhibit consistent differences at certain gene loci, and previous studies have established identification methods based on these differences.

[0005] Random amplified polymorphic DNA (RAPD) or arbitrarily primed polymerase chain reaction (AP-PCR) is a DNA fingerprinting method that has proven to be very useful for strain typing. However, the disadvantages of this technique are that RAPD banding patterns are prone to variability, making replication and interpretation difficult and reproducible. Amplified fragment length polymorphism (AFLP) provides accurate and reproducible strain typing, but the procedure is complex and requires specialized equipment. Both of these DNA fingerprinting methods require the isolation and culture of the pathogen. Subsequently, identification techniques that do not require isolation and culture have emerged, based on the principles of PCR. PCR identification techniques targeting different cell adhesion-related genes, including restriction endonuclease fragment length polymorphism (PCR-RFLP) and high-resolution melting curve analysis (PCR-HRM), have been used to identify MG strains. In addition, core genome multilocus sequence typing (MLST) and mismatch amplification mutation assays (MAMAs) are also widely used in the differential diagnosis of MG. MAMAs offer two methods: one is to analyze the size of PCR products by agarose gel electrophoresis, which has the disadvantage of low sensitivity; the other is to distinguish different PCR amplification products by analyzing the melting temperature (Tm) values ​​of PCR products based on melting curve analysis. This method is simple to operate and has good specificity and sensitivity, but it has certain limitations in clinical sample testing. Currently available identification methods have some shortcomings and cannot achieve quantitative detection. Clinical prevention and control should be analyzed based on the detection ratio of wild-type and vaccine strains, and quantitative detection is needed to assess the severity of wild-type infection and the effectiveness of vaccination.

[0006] Currently, my country relies on vaccination to prevent and control MG infection, with the most widely used attenuated live (MLV) vaccines. Currently approved MLV vaccines in my country include the F, TS-11, and 6 / 85 strains. All three vaccine strains are effective in generating mucosal immunity and protective effects. With the widespread use of live vaccines, the development of differential detection methods to distinguish between vaccine immunity and wild-type infection is essential for evaluating vaccine efficacy and monitoring wild-type infection levels. However, a qualitative and quantitative test method is currently lacking for differential diagnosis of strong and weak MG strains to monitor wild-type infection and the effectiveness of MLV vaccine colonization in vaccinated chickens. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a primer probe designed based on positions 3141 to 3156 of the crmA gene of a wild-type Mycoplasma gallisepticum strain in the preparation of a real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from vaccine strains of Mycoplasma gallisepticum.

[0008] The technical problem that the present invention also aims to solve is to provide a primer-probe combination.

[0009] The technical problem that the present invention also aims to solve is to provide an application of a primer-probe combination in the preparation of a real-time fluorescence quantitative PCR kit for distinguishing wild strains of Mycoplasma gallisepticum from F strains, TS-11 strains, and 6 / 85 strains.

[0010] The final technical problem to be solved by the present invention is to provide a real-time fluorescence quantitative PCR kit that can quantitatively detect and distinguish MG wild-type strains from F strains, TS-11 strains, and 6 / 85 strains.

[0011] Technical solution: In order to solve the above technical problems, the present invention provides the use of primer probes designed based on positions 3141 to 3156 of the crmA gene of wild-type Mycoplasma gallisepticum in the preparation of a real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from vaccine strains of Mycoplasma gallisepticum.

[0012] Among them, when the Mycoplasma gallisepticum vaccine strain is the F strain, positions 2126-2142 of its crmA gene mutate, specifically, position 2131 mutates from T to A, position 2133 mutates from A to C, and position 2137 mutates from G to C.

[0013] Among them, when the Mycoplasma gallisepticum vaccine strain is the F strain, the 3147th position of its crmA gene mutates from T to C, and the 3156th position mutates from G to A; when the Mycoplasma gallisepticum vaccine strain is the TS-11 strain, the 3147th position of its crmA gene mutates from T to G; when the Mycoplasma gallisepticum vaccine strain is the 6 / 85 strain, the 3142-3152 positions of its crmA gene are deleted.

[0014] The present invention also includes a primer probe combination, which includes a primer probe designed based on positions 3141 to 3156 of the crmA gene of a wild strain of Mycoplasma gallisepticum, the primer sequence of the wild strain of Mycoplasma gallisepticum is shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; or / and a primer probe designed based on positions 2126 to 2142 of the crmA gene of the F strain, the primer sequence of the F strain is shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6; or / and a primer probe designed based on positions 518 to 534 of the potC gene of the TS-11 strain, the primer sequence of the TS-11 strain is shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9; or / and a primer probe designed based on positions 1307 to 1322 of the gapA gene of the 6 / 85 strain, the primer sequence of the 6 / 85 strain is shown in SEQ ID NO. ID NO.10 and SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.12.

[0015] The present invention also includes the use of the primer-probe combination in the preparation of a real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from F strains, TS-11 strains, and 6 / 85 strains.

[0016] The present invention also includes a real-time fluorescence quantitative PCR kit for distinguishing wild strains of Mycoplasma gallisepticum from F strains, TS-11 strains, and 6 / 85 strains, which comprises the primer-probe combination.

[0017] The kit further includes a 2×qPCR amplification premix (2×qPCR Probe Master Mix), a negative standard, and a positive standard.

[0018] The positive standard includes a plasmid containing specific detection gene fragments for the MG wild-type strain and three vaccine strains. The sequence of the wild-type strain-specific detection gene fragment is shown in SEQ ID NO. 13, the sequence of the F strain-specific detection gene fragment is shown in SEQ ID NO. 14, the sequence of the TS-11 strain-specific detection gene fragment is shown in SEQ ID NO. 15, and the sequence of the 6 / 85 strain-specific detection gene fragment is shown in SEQ ID NO. 16.

[0019] When used to differentiate MG wild-type strains from F strains, TS-11 strains, and 6 / 85 vaccine strains, follow these steps:

[0020] (1) Extracting total DNA from the sample to be tested;

[0021] (2) Establishment of standard curve for fluorescence quantitative method 8 copies / μL to 10 1 The copies / μL serial dilutions were used as templates, and fluorescent quantitative PCR amplification was performed with the primers and probes to establish a standard curve;

[0022] (3) using the total DNA extracted in step (1) as a template and performing fluorescent quantitative PCR amplification using the primer-probe combination;

[0023] (4) Automatically detect and collect fluorescence signals during the PCR reaction, and make qualitative judgments based on the CT value. The judgment criteria are: CT value > 37 or no specific fluorescence curve is judged as negative, 35 ≤ CT value ≤ 37 is judged as suspicious, 30 ≤ CT value < 35 is judged as weak positive, and CT value < 30 is judged as positive;

[0024] (5) The CT value read in step (4) can be used to calculate the copy number based on the standard curve to achieve quantitative detection.

[0025] Among them, the PCR amplification system of step (2) includes 2×qPCR Probe Master Mix, a primer-probe combination, wherein the primers include MG wild virus identification detection PCR upstream primer and any one of the MG vaccine strain identification detection PCR upstream primers, MG wild virus identification detection PCR downstream primer and the corresponding MG vaccine strain identification detection PCR downstream primer, MG wild virus detection probe and the corresponding vaccine strain identification detection probe, the primer concentration is 2-10 μM, the probe concentration is 1-5 μM, DNA template, and sterilized water.

[0026] Wherein, the PCR amplification system of step (2) is 20 μL; the system contains 10 μL 2×qPCR Probe MasterMix, 2 μL primer / probe combination, wherein the primer / probe combination includes 4 μM MG wild virus identification detection PCR upstream primer (MG-W-Uni-F) and any one of the MG vaccine strain identification detection PCR upstream primers (MG-F36-F, MG-TS11-F, MG-6 / 85-F), 4 μM MG wild virus identification detection PCR downstream primer (MG-W-Uni-R) and the corresponding MG vaccine strain identification detection PCR downstream primer (MG-F36-R, MG-TS11-R, MG-6 / 85-R), 2 μM MG wild virus detection probe (MG-W-Uni-P) and the corresponding vaccine strain detection probe (MG-F36-P, MG-TS11-P, MG-6 / 85-PV), 2 μL DNA template and sterilized water to make up to 20 μL.

[0027] Preferably, the reaction conditions are: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, 40 cycles; collecting the fluorescence signal at 60°C for 30 s (the program needs to set up dual fluorescence channels of FAM and VIC).

[0028] Among them, it is recommended to prepare the identification detection system according to n+1+1+1, where n is the number of test samples, 1≤n≤93, and 1 represents negative quality control, wild virus positive quality control and corresponding vaccine virus positive quality control.

[0029] Prepare the corresponding number of reaction systems according to the above system, centrifuge briefly and then dispense into 0.2mL fluorescent quantitative PCR tubes, 18μL per tube, move to the nucleic acid extraction area for sampling, add 2μL of DNA template sample to the sample test tube, add 2μL of the corresponding positive plasmid to the positive quality control reaction tube, make the corresponding mark and centrifuge briefly for detection.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the kit of the present invention has high sensitivity, strong specificity, and good repeatability, and can accurately realize the identification and detection of MG wild-type strains and vaccine strains (F strain, TS-11 strain, 6 / 85 strain) at the same time. It can be used to identify MG isolates that have been separated, cultured and purified, and can also be used to evaluate the in vivo replication efficiency of attenuated live vaccines (F strain, TS-11 strain, 6 / 85 strain) in immunized chickens and monitor wild-type virus infection, achieving rapid and accurate detection while ensuring specificity and sensitivity; it can simultaneously realize qualitative and quantitative identification and detection of MG wild-type strains and vaccine strains; it can be used for the identification and detection of MG strains that have been separated and purified, and can also be used for the identification and detection of wild-type strains and vaccine strains coexisting in clinical samples.

[0031] In addition, four sets of PCR primers targeting wild-type strains and three vaccine strains can be used in combination with MGB-Taqman probes. The present invention demonstrates that the wild-type virus detection system can be combined with the three vaccine strain detection systems for dual qPCR detection. The test results show that dual qPCR can simultaneously realize the identification and detection of wild-type strains and vaccine strains with good sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Design sites for specific identification and detection of MG wild-type strains by primers and probes;

[0033] Figure 2 Design sites for F strain-specific identification and detection primers and probes;

[0034] Figure 3 Design sites for TS-11 strain-specific identification and detection primers and probes;

[0035] Figure 4Designed sites for 6 / 85 strain-specific identification detection primers and probes;

[0036] Figure 5 This is the fluorescence curve of the MG wild-type virus and three vaccine strains amplified by the MG wild-type virus specific detection system in the specificity test;

[0037] Figure 6 This is the fluorescence curve of the MG wild-type strain and three vaccine strains amplified by the MG vaccine (F strain) specificity detection system in the specificity test;

[0038] Figure 7 This is the fluorescence curve of the MG wild-type strain and three vaccine strains amplified by the MG vaccine (TS-11 strain) specificity detection system in the specificity test;

[0039] Figure 8 This is the fluorescence curve of the MG wild-type strain and three vaccine strains amplified by the MG vaccine (6 / 85 strains) specificity detection system in the specificity test;

[0040] Figure 9 This is the amplification curve of the MG wild-type virus-specific detection system for the corresponding positive standard diluted in series;

[0041] Figure 10 The standard curve is drawn based on the amplification results of the corresponding positive standard diluted in series according to the MG wild-type virus strain specific detection system;

[0042] Figure 11 This is the amplification curve of the MG F strain-specific detection system for the corresponding positive standard diluted in series;

[0043] Figure 12 The standard curve is drawn based on the amplification results of the corresponding serially diluted positive standards according to the MG F strain-specific detection system;

[0044] Figure 13 This is the amplification curve of the MG TS-11 strain-specific detection system for the corresponding positive standard diluted in series;

[0045] Figure 14 The standard curve is drawn based on the amplification results of the corresponding serially diluted positive standards according to the MG TS-11 strain-specific detection system;

[0046] Figure 15 This is the amplification curve of the MG 6 / 85 strain-specific detection system for the corresponding positive standard with serial dilutions;

[0047] Figure 16 The standard curve is drawn based on the amplification results of the corresponding serially diluted positive standards according to the 6 / 85 strain-specific detection system;

[0048] Figure 17This is the amplification curve of the wild-type F strain-specific probe / primer mixture identification detection system under the serial dilution of the corresponding positive standard in the dual qPCR test of wild-type and vaccine strains (FAM and VIC fluorescence signals are collected simultaneously);

[0049] Figure 18 This is the amplification curve of the wild-type and vaccine strain dual qPCR test using the wild-type and TS-11 strain-specific probe / primer mixture identification detection system for the corresponding serial dilutions of the positive standard (FAM and VIC fluorescence signals were collected simultaneously);

[0050] Figure 19 This is the amplification curve of the wild-type 6 / 85 strain-specific probe / primer mixture identification detection system under the serial dilution of the corresponding positive standard in the dual qPCR test of wild-type and vaccine strains (FAM and VIC fluorescence signals were collected simultaneously);

[0051] Figure 20 This is the amplification curve of the MG wild-type F strain specific probe / primer mixture identification detection system (Group 1) on some clinical sample DNA;

[0052] Figure 21 This is the amplification curve of the MG wild-type TS-11 strain-specific probe / primer mixture identification and detection system (Group 2) on some clinical sample DNA. DETAILED DESCRIPTION

[0053] The following detailed and comprehensive description of the embodiments of the present invention is provided in conjunction with specific examples. Obviously, the described examples are only intended to illustrate a portion of the present invention and should not be construed as limiting the scope of the invention. In the examples, the specific conditions not otherwise specified are all carried out according to conventional conditions or manufacturer's recommendations. Reagents or instruments used that do not indicate the manufacturer are all commercially available conventional products.

[0054] Example 1

[0055] 1. Primer and probe design

[0056] According to the sequence comparison results of the NCBI database of MG wild-type strains and vaccine strains (F strain, TS-11 strain, 6 / 85 strain), F strain, TS-11 strain and 6 / 85 strain all had different forms of mutations at positions 3141-3156 of the crmA gene (NCBI accession number: AE015450.2) relative to the MG wild-type strain. Among them, the F strain had two point mutations, with position 3147 mutated from T to C and position 3156 mutated from G to A. The TS-11 strain had one site mutation, with position 3147 mutated from T to G. The 6 / 85 strain had a deletion at positions 3142-3152. Further comparison showed that the crmA of the F strain The above three point mutations occurred at positions 2126-2142 of the gene (NCBI accession number: CP001873.1), specifically, position 2131 mutated from T to A, position 2133 mutated from A to C, and position 2137 mutated from G to C; the potC gene of the TS-11 strain (NCBI accession number: NZ_CP044225.1) mutated at positions 518-534, specifically, position 526 mutated from C to G; the gapA gene (NCBI accession number: NZ_CP044224.1) of 6 / 85 strains mutated at positions 1307-1322, specifically, position 1316 mutated from A to G.

[0057] Upstream and downstream primers were designed in the above-mentioned regions of the crmA gene of the MG wild-type strain, the crmA gene of the F strain, the potC gene of the TS-11 strain, and the gapA gene of the 6 / 85 strain, respectively, and were named MG-W-Uni-F (SEQ ID NO.1), MG-W-Uni-R (SEQ ID NO.2); MG-F36-F (SEQ ID NO.4), MG-F36-R (SEQ ID NO.5); MG-TS11-F (SEQ ID NO.7), MG-TS11-R (SEQ ID NO.8); MG-6 / 85-F (SEQ ID NO.10), MG-6 / 85-R (SEQ ID NO.11).

[0058] Specific probes were designed based on the specific mutation sites in the above genes of MG wild strains and vaccine strains (F strain, TS-11 strain, 6 / 85 strain) (specific identification detection primers for MG wild strains and three vaccine strains, probe design sites are shown in Figure 1-4)MG-W-Uni-P (SEQ ID NO. 3), MG-F36-P (SEQ ID NO. 6), MG-TS11-P (SEQ ID NO. 9), and MG-6 / 85-PV (SEQ ID NO. 12). The MG-W-Uni-P probe is labeled with a reporter group FAM at its 5' end and a fluorescence quencher MGB at its 3' end; the MG-F36-P, MG-TS11-P, and MG-6 / 85-PV probes are labeled with a reporter group VIC at their 5' end and a fluorescence quencher MGB at their 3' end. Primer and probe designs are shown in Table 1.

[0059] Table 1. Primer and probe sequences

[0060]

[0061]

[0062] 2. Specificity test

[0063] The MG wild-type strain Rlow (strain number: CVCC1651) was purchased from the China Veterinary Microbiological Culture Collection Center, the F strain was purchased from Qingdao Yibang Bioengineering Co., Ltd., the TS-11 vaccine strain was purchased from Australian Bioresources Company, and the 6 / 85 vaccine strain was purchased from Intervet USA Branch. Genomic DNA was extracted from pure cultures of the Rlow, F, TS-11, and 6 / 85 strains, respectively. qPCR experiments were performed on DNA templates of wild-type strains and three vaccine strains (F, TS-11, and 6 / 85) using four specific detection systems (wild-type, F, TS-11, and 6 / 85) to verify the specificity of this fluorescence quantitative method. The qPCR reaction system was 20 μL (optimized reaction system using the Novozymes AceQ qPCR Probe Master Mix kit). Taking the wild-type specific detection system as an example, the identification system contained 10 μL 2×qCR Probe Master Mix, 4 μM wild-type specific detection PCR upstream primer (MG-W-Uni-F), 4 μM wild-type specific detection PCR downstream primer (MG-W-Uni-R), 2 μM wild-type specific detection MGB probe (MG-W-Uni-P), and 2 μL template DNA, and sterilized water was added to 20 μL. The reaction conditions were: 95°C for 5 minutes; 95°C for 10 seconds, 60°C for 30 seconds, for 40 cycles (Novagen AceQ qPCR Probe Master Mix Kit optimized reaction system); signal collection at 60°C for 30 seconds. The wild-type MG strain genome and the genomes of three vaccine strains (F strain, TS-11 strain, and 6 / 85 strain) were used as templates for testing. The wild-type strain-specific detection system test results showed that specific fluorescence signals could be detected only when wild-type strain DNA was used as a template. The results are shown in Figure 2. Figure 5 The results of the F strain identification and detection system test showed that specific fluorescence signals could be detected only when the F strain DNA was used as a template. Figure 6 ; The test results of the TS-11 strain identification detection system showed that specific fluorescence signals could be detected only when TS-11 strain DNA was used as a template. Figure 7 The test results of the 6 / 85 strain identification detection system showed that specific fluorescence signals could be detected only when the 6 / 85 strain DNA was used as a template. Figure 8 The above results indicate that the real-time fluorescence quantitative PCR method for distinguishing MG wild-type strains from vaccine strains (F strain, TS-11 strain, 6 / 85 strain) established in the present invention has good specificity and can accurately distinguish and diagnose pure cultured MG wild-type strains from vaccine strains.

[0064] Example 2

[0065] 1. Preparation of positive standards for real-time fluorescence quantitative PCR

[0066] Primers and probes were designed based on the results of laboratory sequence alignment and the specific gene sites of MG wild-type and vaccine strains reported in the literature. Genomic DNA of the corresponding MG strain was used as a template, and the target fragments were amplified using the primers of each reaction system. After the fragment sizes were correctly identified, they were cloned into pEASY-T3 Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd., catalog number: CT301) and transformed into transⅠ-T1 competent cells (Quanshijin, catalog number: CD501-02). Plasmids were extracted using a high-purity plasmid mini-preparation kit (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW0500M) for use as positive standards (T3 vector plasmid containing specific detection gene fragments, wherein the sequence of the wild-type specific detection gene fragment is shown in SEQ ID NO.13, the sequence of the F strain specific detection gene fragment is shown in SEQ ID NO.14, the sequence of the TS-11 strain specific detection gene fragment is shown in SEQ ID NO.15, and the sequence of the 6 / 85 strain specific detection gene fragment is shown in SEQ ID NO.16). The T3 vector sequence (full version, catalog number: CT301) can be found on the official website.

[0067] The primer sequences for amplifying a portion of the crmA gene of the MG wild-type strain are shown in SEQ ID NOs. 1 and 2. The primer sequences for amplifying a portion of the crmA gene of the F strain are shown in SEQ ID NOs. 4 and 5. The primer sequences for amplifying a portion of the potC gene of the TS-11 strain are shown in SEQ ID NOs. 7 and 8. The primer sequences for amplifying a portion of the gapA gene of the 6 / 85 strain are shown in SEQ ID NOs. 10 and 11.

[0068] 2. Establishment of standard curve and sensitivity test of real-time fluorescence quantitative PCR method

[0069] The positive standard plasmids corresponding to the wild-type strain and vaccine strains (F strain, TS-11, 6 / 85) were extracted from the cloned transⅠ-T1 competent cells using a high-purity plasmid extraction kit (Jiangsu Kangwei Century Biotechnology Co., Ltd., catalog number: CW0500M). The plasmid concentration was measured using a spectrophotometer (NanoDrop2000, Thermo Fisher Scientific, USA) according to the formula = (C × 10 -9 ) / (M×660)×6.02×10 23 (C is the concentration of the standard, M is the number of bases in the constructed plasmid) Calculate the number of plasmid copies. 8A 10-fold serial dilution from 1 copy / μL to 1 copy / μL was performed as a template to establish a standard curve for the real-time fluorescence quantitative PCR method. Real-time fluorescence quantitative PCR amplification was performed according to the preferred conditions in Example 1. The PCR reagent used in the experiment was 2×AceQ qPCR Probe Master Mix (Nanjing Novozyme Biotechnology Co., Ltd., product number: Q112-02 / 03). The amplification curve of the wild-type positive standard and the drawn standard curve are shown in Figure 2. Figure 9 and Figure 10 The standard curve of the wild strain specific identification system is y = -3.27x + 39.28, R2 = 1, and the amplification efficiency is 102.06%; the amplification curve of the F strain positive standard and the drawn standard curve are as follows Figure 11 and Figure 12 The standard curve of the F strain specific identification system is y = -3.32x + 39.6, R2 = 0.999, and the amplification efficiency is 100.05%; the amplification curve of the TS-11 strain positive standard and the drawn standard curve are as follows Figure 13 and Figure 14 The standard curve of the TS-11 strain specific identification system is y = -3.45x + 39.6, R2 = 0.999, and the amplification efficiency is 95.02%; the amplification curve of the 6 / 85 strain positive standard and the drawn standard curve are shown in Figure 15 Figure 16 The standard curve of the 6 / 85 strain-specific identification system was y = -3.21x + 38.18, R2 = 0.999, and the amplification efficiency was 105.01%.

[0070] As can be seen from the figure, the detection sensitivity of the real-time fluorescence quantitative PCR method for the positive standards of wild virus and each vaccine strain can reach 10 1 The minimum detection limit CT range of each identification system is between 35-37. Taking into account the problem of cross contamination of clinical samples, this invention defines this range as suspected positive. CT values ​​<35 are judged as positive (CT values ​​between 30-35 can be judged as weak positive, and sample CT values ​​≤10 2 copies / μL; CT value <30 can be judged as positive, and the sample CT value >10 2 copies / μL).

[0071] 3. Repeatability test

[0072] Using three different concentrations of positive standards as templates, intra-group and inter-group parallel tests were performed. Intra-group parallel testing involves performing three replicates of each sample in a single PCR test; inter-group parallel testing involves performing three different batches of each sample. The intra-group and inter-group coefficients of variation were calculated to analyze the repeatability of the test. The test results showed that both the intra-group and inter-group coefficients of variation of this fluorescence quantitative PCR detection method were less than 0.3%, demonstrating excellent repeatability and stability. The corresponding repeatability test results for the MG wild-type strain identification system are shown in Table 2, the corresponding repeatability test results for the F strain identification system are shown in Table 3, the corresponding repeatability test results for the TS-11 strain identification system are shown in Table 4, and the corresponding repeatability test results for the 6 / 85 strain identification system are shown in Table 5.

[0073] Table 2. Repeatability test of MG wild strain detection

[0074]

[0075] Table 3. Repeatability test of MG F strain detection

[0076]

[0077]

[0078] Table 4. Repeatability test of MG TS-11 vaccine strain

[0079]

[0080] Table 5. Repeatability test of MG 6 / 85 vaccine strain

[0081]

[0082] 4. Dual fluorescence quantitative PCR test of wild-type and vaccine strains

[0083] The wild virus identification detection system was combined with the three vaccine identification detection systems to form a dual fluorescence quantitative PCR detection system for mixed testing. The wild virus (Rlow strain) whole genome DNA and the corresponding vaccine strain whole genome DNA were used as templates for testing. The initial concentration of the whole genome DNA was uniformly set at 0.6 ng / μL, and it was tested in a gradient dilution (the highest dilution was 10 7The reaction system was 20 μL, containing 10 μL 2× qPCR Probe Master Mix, 4 μM MG wild-type virus identification and detection PCR upstream primer (MG-W-Uni-F) and 4 μM of any one of the MG vaccine strain identification and detection PCR upstream primers (MG-F36-F, MG-TS11-F, MG-6 / 85-F), 4 μM MG wild-type virus identification and detection PCR downstream primer (MG-W-Uni-R) and 4 μM of the corresponding MG vaccine strain identification and detection PCR downstream primer (MG-F36-R, MG-TS11-R, MG-6 / 85-R), 2 μM MG wild-type virus detection probe (MG-W-Uni-P) and 2 μM of the corresponding vaccine strain detection probe (MG-F36-P, MG-TS11-P, MG-6 / 85-PV), 2 μL DNA template, and sterile water to 20 μL. The reaction conditions were: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, 40 cycles; fluorescence signal was collected at 60°C for 30 s (the program required the setting of FAM and VIC dual fluorescence channels). The results showed that dual fluorescence quantitative PCR had high sensitivity and could detect 10 7 The results of the double fluorescence quantitative PCR test of the wild-type F strain are as follows: Figure 17 ; The results of the dual fluorescence quantitative PCR test of wild virus-TS-11 vaccine strain are as follows Figure 18 ; The results of the dual fluorescence quantitative PCR test of the wild-type 6 / 85 vaccine strain are as follows Figure 19 .

[0084] The optimal reaction system was determined by adjusting the primer and probe concentrations. Appropriate ratios were increased or decreased according to the primer:probe ratio recommended by the Norvegian qPCR Probe Master Mix (primer:probe ratio, 2:1). A 20-μL system contained 10 μL of 2× qPCR Probe Master Mix. Three replicates were added with three primer concentrations (2 μM, 6 μM, and 10 μM) and three probe concentrations (1 μM, 3 μM, and 5 μM). Two μL of DNA template was added, and the volume was made up to 20 μL with sterile water. The reaction conditions were: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Fluorescence signal was collected at 60°C for 30 s (the program required dual fluorescence channels for FAM and VIC). The test results show that when the primer and probe concentrations are 2μM and 1μM, respectively, identification detection can still be achieved, but the CT value is lower than that of the medium and high concentration test groups (the primer and probe concentrations are 6μM, 3μM and 10μM, 5μM, respectively), which proves that its detection efficiency is reduced. No significant difference was found in the CT values ​​detected between the medium and high concentration test groups, only the fluorescence intensity was different. Among them, the fluorescence intensity of the medium concentration test group can meet the detection requirements. Taking into account factors such as clinical use costs, the present invention ultimately determines that the optimal concentrations of MG wild-type-vaccine strain dual fluorescence PCR primers and probes with a total volume of 20μL are 4μM and 2μM. In production practice, personnel in the relevant technical field can adjust the concentrations of primers and probes in the dual fluorescence quantitative PCR system according to needs. It can be determined that the primer concentration in the 20μL system can achieve identification detection in the range of 2μM-10μM, and the probe concentration can achieve identification detection in the range of 1μM-5μM.

[0085] Table 6 Test of optimal primer and probe concentrations in dual fluorescence quantitative PCR system

[0086]

[0087] Example 3 Application of a Real-Time Fluorescence Quantitative PCR Kit for Identifying Wild-Type Virus Strains and Vaccine Strains (F Strain, ts-11, 6 / 85) in Clinical Sample Detection

[0088] The present invention has been verified in the laboratory to be able to detect the DNA of pure cultures of MG wild strains and three vaccine strains, and then a large number of clinical samples are tested. These samples come from different regions, including Jiangsu, Shandong, Hebei and other places. Different farms use different MG attenuated live vaccines. Therefore, we select different wild strain-vaccine strain dual fluorescence quantitative PCR combinations for detection, and further optimize the reaction system. The different combinations of identification detection primers and probes are mixed in proportion to prepare a nucleic acid mixture, which is convenient for the experimenter to operate and prevent contamination caused by repeated operations. Among them, the wild strain-F strain identification detection kit (taking 100 reactions as an example) contains 1000 μL qPCR MiX, 200 μL nucleic acid mixture (wild strain and F strain specific upstream and downstream primers are both 400 μM, wild strain and F strain specific identification probes are both 200 μM), 600 μL sterile water, 40 μL negative quality control (plasmid solution without specific target fragment), and 40 μL positive quality control (plasmid solution containing specific target fragment). The reagents contained in the Wild-type TS-11 and Wild-type 6 / 85 Identification Detection Kit (100 reactions) are generally the same as those in the Wild-type F Identification Detection Kit. The probe mixture in the Wild-type TS-11 Identification Detection Kit consists of 400 μM of wild-type and TS-11-specific upstream and downstream primers and 200 μM of the wild-type and TS-11-specific identification probe. The probe mixture in the Wild-type 6 / 85 Identification Detection Kit consists of 400 μM of wild-type and 6 / 85-specific upstream and downstream primers and 200 μM of the wild-type and 6 / 85-specific identification probe.

[0089] The optimized reaction system was used to test clinical samples, with a total of 540 clinical samples tested, including 60 clinical samples of immune F strain, 480 clinical samples of immune TS-11 vaccine strain, and 0 clinical samples of immune 6 / 85 vaccine strain (rarely used in clinical practice). The optimized dual fluorescence quantitative PCR system was used to test the samples (including nucleic acid mixture system). A total of 60 clinical samples of immune F strain were tested, of which 11 were positive for wild-type strain and 11 for F strain (tested using wild-type strain-F strain nucleic acid mixture system). The amplification curves of some clinical samples of immune F strain are shown as follows: Figure 20 A total of 480 clinical samples were immunized with the TS-11 strain, of which 128 were positive for the wild strain and 48 were positive for the TS-11 strain (tested using a wild strain-TS-11 strain nucleic acid mixture system). The amplification curves of some clinical samples immunized with the TS-11 strain are shown in Figure 2. Figure 21The test results demonstrate that the present invention can detect and identify MG clinical samples. The optimized detection system (including the nucleic acid mixture) showed no significant difference in detection results compared to the original dual fluorescence quantitative PCR detection system, demonstrating its applicability to clinical sample testing. Therefore, the final form of the present invention provides a nucleic acid mixture for detection and identification, along with other components, the specific components being the same as described above.

Claims

1. Based on wild strains of Mycoplasma gallisepticum crmA The invention discloses an application of primer probes designed at positions 2126-2142 and 3141-3156 of a gene in preparing a real-time fluorescence quantitative PCR kit for distinguishing wild strains of Mycoplasma gallisepticum from vaccine strains of Mycoplasma gallisepticum, wherein the vaccine strains of Mycoplasma gallisepticum are F strain, TS-11 strain and 6 / 85 strain. When the vaccine strain of Mycoplasma gallisepticum is F strain, the real-time fluorescence quantitative PCR kit is used for distinguishing wild strains of Mycoplasma gallisepticum from vaccine strains of Mycoplasma gallisepticum. crmA The 2126-2142 positions of the gene mutated, specifically the 2131st position mutated from T to A, the 2133rd position mutated from A to C, and the 2137th position mutated from G to C. When the Mycoplasma gallisepticum vaccine strain is the F strain, crmA The 3147th position of the gene mutates from T to C, and the 3156th position mutates from G to A; when the Mycoplasma gallisepticum vaccine strain is TS-11 strain, crmA The 3147th position of the gene mutates from T to G; when the Mycoplasma gallisepticum vaccine strain is 6 / 85 strains, crmA There is a deletion at positions 3142-3152 of the gene.

2. A primer-probe combination, characterized in that: The primer probe combination includes a wild strain of Mycoplasma gallisepticum crmA The primers and probes designed at positions 2126-2142 and 3141-3156 of the gene, the primer sequences of the wild strain of Mycoplasma gallisepticum are shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; or / and based on the F strain crmA The primers and probes designed for positions 2126-2142 of the gene, the primer sequences of the F strain are shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6; or / and based on the TS-11 strain potC The primers and probes designed for positions 518-534 of the gene, the primer sequences of the TS-11 strain are shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9; or / and based on the 6 / 85 strain gapA The primers and probes designed for positions 1307-1322 of the gene, the primer sequences of the 6 / 85 strains are shown in SEQ ID NO.10 and SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.

12.

3. Use of the primer-probe combination according to claim 2 in the preparation of a real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from F strains, TS-11 strains, and 6 / 85 strains.

4. A real-time fluorescence quantitative PCR kit for distinguishing wild-type Mycoplasma gallisepticum strains from F strains, TS-11 strains, and 6 / 85 strains, characterized in that: It comprises the primer-probe combination according to claim 2.

5. The kit according to claim 4, characterized in that The kit also includes a 2×qPCR amplification premix and a positive standard.

6. The kit according to claim 4, wherein The positive standard comprises a plasmid containing specific detection gene fragments of MG wild strain and three vaccine strains.

7. The kit according to claim 5 or 6, characterized in that To differentiate wild-type MG strains from F strains, TS-11 strains, and 6 / 85 vaccine strains, follow these steps: (1) Extract the total DNA of the sample to be tested; (2) Establishment of standard curve for fluorescence quantitative method: The positive standard plasmid was 8 copies / μL to 10 1 The copies / μL serial dilutions were used as templates, and fluorescent quantitative PCR amplification was performed with the primers and probes described in claim 4 to establish a standard curve; (3) using the total DNA extracted in step (1) as a template, and performing fluorescent quantitative PCR amplification using the primer-probe combination described in claim 4; (4) Automatically detect and collect fluorescence signals during the PCR reaction, and make qualitative judgments based on the CT value. The judgment criteria are: CT value > 37 or no specific fluorescence curve is judged as negative, 35 ≤ CT value ≤ 37 is judged as suspicious, 30 ≤ CT value < 35 is judged as weakly positive, and CT value < 30 is judged as positive; (5) The CT value read in step (4) can be used to calculate the copy number based on the standard curve to achieve quantitative detection.

8. The kit according to claim 7, characterized in that The PCR amplification system of step (2) includes 2×qPCRProbe Master Mix, a primer-probe combination, wherein the primers include an MG wild virus identification detection PCR upstream primer and any one of the MG vaccine strain identification detection PCR upstream primers, an MG wild virus identification detection PCR downstream primer and the corresponding MG vaccine strain identification detection PCR downstream primer, an MG wild virus detection probe and the corresponding vaccine strain identification detection probe, the primer concentrations are all 2~10μM, the probe concentrations are all 1~5μM, a DNA template, and sterilized water.

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