QPCR (quantitative polymerase chain reaction) rapid detection method for mycoplasma ovipneumoniae

Abstract

The TaqMan fluorescent quantitative PCR method for detecting the mycoplasma ovipneumoniae, provided by the invention, has very high specificity, sensitivity and stability, can be used for screening and detecting mycoplasma ovipneumoniae positive samples in various clinical samples, and has the characteristics of simplicity and convenience in operation, short detection period and high detection efficiency.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a TaqMan fluorescent quantitative PCR method for Mycoplasma ovipneumoniae. Background Art

[0002] Mycoplasma ovipneumoniae (MO) is the main pathogen causing mycoplasma pneumonia of goats and sheep (MPGS). MPGS is highly contagious, mainly through respiratory infection, and can also be transmitted vertically. Sick sheep and resistant sheep are the main sources of infection. Sick sheep are characterized by chronic non-progressive pneumonia, and clinically manifested mainly by coughing, wheezing, runny nose, anemia, emaciation, growth retardation and other symptoms. The disease is widely prevalent in sheep-raising countries, causing serious economic losses to the sheep industry.

[0003] At present, the methods commonly used to detect Mycoplasma ovipneumoniae are mainly the color change unit test method (CCU method) and the plate count method (CFU method). Compared with the CCU method, the CFU method has more stringent culture conditions for Mycoplasma ovipneumoniae, but it can more intuitively observe the colony morphology of Mycoplasma ovipneumoniae and calculate the number of Mycoplasma ovipneumoniae. However, both methods have long detection cycles, cumbersome detection steps, and only rely on naked eye observation when judging the results. There is no sufficient reliable basis, which affects the accuracy of the experiment and limits the promotion and application of Mycoplasma ovipneumoniae in rapid detection.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a new qPCR rapid detection method for Mycoplasma ovipneumoniae.

[0006] The first object of the present invention is to provide a primer set for detecting Mycoplasma ovipneumoniae, the primer set comprising an upstream primer and a downstream primer, the upstream primer sequence is shown in SEQ ID NO.4 or SEQ ID NO.6; the downstream primer sequence is shown in SEQ ID NO.5 or SEQ ID NO.7.

[0007] Furthermore, the primer set also includes a probe, and the probe sequence is shown in SEQ ID NO.10 or SEQ ID NO.11.

[0008] Preferably, the primer set is an upstream primer with a sequence as shown in SEQ ID NO.4, a downstream primer with a sequence as shown in SEQ ID NO.5 and a probe with a sequence as shown in SEQ ID NO.10.

[0009] Furthermore, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0010] Furthermore, the fluorescent reporter group includes a FAM group, a CY5 group or a HEX group; the fluorescent quencher group includes a MGB group, a BHQ1 group or a BHQ2 group.

[0011] Preferably, the fluorescent reporter group is a FAM group, and the fluorescent quencher group is a MGB group.

[0012] The second object of the present invention is to provide a method for detecting Mycoplasma ovipneumoniae for non-disease diagnosis and treatment purposes, using a qPCR reaction system including the above-mentioned detection primers to amplify and detect samples containing Mycoplasma ovipneumoniae.

[0013] The detection method has good specificity and sensitivity to Mycoplasma ovipneumoniae and can perform qualitative and / or quantitative detection on the number of live bacteria and the amount of total protein of Mycoplasma ovipneumoniae.

[0014] The specific qualitative evaluation process is as follows:

[0015] In qualitative detection, a qPCR reaction system and reaction procedure including the above primer probe combination are used to amplify and detect samples that may contain Mycoplasma ovipneumoniae. When the test sample has a CT value, the test result is positive, and it can be determined that the sample contains Mycoplasma ovipneumoniae; when the test sample has no CT value, the test result is negative, and it can be determined that the sample does not contain Mycoplasma ovipneumoniae.

[0016] The method can quantify the number of viable bacteria in the sample to be tested, and the specific method steps are: a) using a standard of known concentration for gradient dilution to obtain standard solutions of multiple concentrations, using the above-mentioned qPCR reaction system and reaction program to amplify the standard solutions of each concentration and draw a standard curve; b) according to the established standard curve, calculating the copy number of the sample to be tested; c) substituting the copy number into the regression equation, lgCFU=0.8032×lgcopy+1.0377, to calculate the number of viable bacteria in the sample.

[0017] The method can also quantify the total protein amount in the sample to be tested, and the specific method steps are: a) using a standard of known concentration for gradient dilution to obtain standard solutions of multiple concentrations, using the above-mentioned qPCR reaction system and reaction procedure to amplify the standard solutions of each concentration and draw a standard curve; b) according to the established standard curve, calculate the copy number of the sample to be tested; c) substituting the copy number into the regression equation, total protein amount = 70.832×lgcopy-549.74, and calculating the total protein amount of the sample.

[0018] In the above method steps, the gradient dilution of the standard sample adopts a 10-fold gradient dilution; the "lgcopy" in the regression equation is the logarithm of the copy number of the sample to be tested.

[0019] In some optional embodiments, the working concentration of the upstream primer is 0.12 to 0.44 μM, preferably 0.28 μM;

[0020] The working concentration of the downstream primer is 0.12-0.44 μM, preferably 0.28 μM;

[0021] The working concentration of the probe is 0.02-0.18 μM, preferably 0.10 μM.

[0022] It should be noted that, in the present invention, the working concentrations of primers and probes refer to the concentrations of primers and probes in the amplification reaction system.

[0023] In the present invention, the primer pairs and probe concentrations in the real-time fluorescence quantitative PCR reaction system are further optimized and adjusted, which is more conducive to the PCR reaction.

[0024] Preferably, the qPCR reaction system further comprises Premix Ex Taq, a DNA template and deionized water.

[0025] Furthermore, the qPCR amplification reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s; annealing at 54.8-60.2°C for 30 s; extension at 72°C for 20 s, with a cycle number of 35-45.

[0026] Preferably, the amplification reaction procedure is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s; annealing at 57.2°C for 30 s; extension at 72°C for 20 s, with 40 cycles.

[0027] The third object of the present invention is to provide a kit for detecting Mycoplasma ovipneumoniae, the kit comprising: a qPCR reaction system containing the above-mentioned detection primer set.

[0028] Furthermore, the kit also includes a positive control substance and / or a negative control substance.

[0029] Preferably, the positive control substance is a recombinant plasmid containing a Mycoplasma ovipneumoniae gene, and the negative control substance is deionized water.

[0030] The fourth object of the present invention is to provide the use of the above kit in the qualitative or quantitative detection of Mycoplasma ovipneumoniae.

[0031] Beneficial effects of the present invention:

[0032] (1) The primers and probes of the present invention have good sensitivity and specificity to Mycoplasma ovipneumoniae and can effectively detect Mycoplasma ovipneumoniae.

[0033] (2) The detection method of the present invention can perform qualitative and quantitative detection of Mycoplasma ovipneumoniae. The presence or absence of a CT value in the sample to be tested can be used to determine whether the sample contains Mycoplasma ovipneumoniae. At the same time, the number of viable bacteria and the amount of total protein in the sample to be tested can be calculated according to the regression equation.

[0034] (3) The detection method of the present invention has good specificity, repeatability and stability, with a minimum detection limit of 2.43 copies / μL, high sensitivity, short detection cycle, accurate quantitative determination of viable counts and total protein, and can effectively solve the problems of long detection cycle, low detection efficiency and inaccurate detection results of existing quantitative bacterial concentration methods (CCU method and CFU method) of Mycoplasma ovipneumoniae. The method established by the present invention is of great significance for the rapid quantitative detection of Mycoplasma ovipneumoniae cultures and vaccine semi-finished products, and the detection and promotion of this method can also provide a reference for further understanding of the genetic evolution of Mycoplasma ovipneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 is the amplification result of primers (DnaG-F, DnaG-R), where 1-2 are Mycoplasma ovipneumoniae strains, 3-4 are recombinant plasmid PUC57-DnaG, and 5 is a negative control;

[0037] Figure 2 1 is the amplification curve of the six primer-probe combinations in Example 1 when detecting the same PUC57-DnaG sample;

[0038] Figure 3 2.43×10 in Example 5 9 ~2.43×10 2 Amplification curves of the concentration gradient standard of copies / μL, A, B and C are the amplification curves of the first combination, the second combination and the fourth combination respectively;

[0039] Figure 41 to 3 are amplification curves of different pathogens in Example 8, wherein 1 to 3 are Mycoplasma ovipneumoniae, 4 to 15 are Mycoplasma capricolum 87001, Mycoplasma capricolum subsp. capricolum, Mycoplasma mycoides subsp. capricolum PG3, Mycoplasma agalactiae PG2, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Escherichia coli, Pasteurella, Mannheimia, Haemophilus parasuis HPS, and Staphylococcus aureus;

[0040] Figure 5 The results of the common PCR method in Example 9 for 113 clinical samples, where M: 2000 DNA marker; 1-30: nasal swab; 31-113: lung tissue; -: negative control; +: positive control;

[0041] Figure 6 The results of the conventional PCR amplification and identification of the three strains of Mycoplasma ovipneumoniae isolated and purified from the lung tissue in Example 9; wherein M: 2000 DNA marker; 1-4: isolate-1; 5-8: isolate-2; 9-12: isolate-3; 13: culture medium; -: negative control; +: positive control;

[0042] Figure 7 This is the linear correlation between the total protein content and viable count of Mycoplasma ovipneumoniae in Example 10. DETAILED DESCRIPTION

[0043] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0044] The main reagents and their manufacturers used in the following examples are shown in Table 1:

[0045] Table 1 Main reagents and their manufacturers

[0046]

[0047] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0048] Example 1 Screening of primers and probes

[0049] DnaG belongs to Toprim_N superfamily, is a DNA-dependent RNA polymerase, for synthesizing short-chain RNA initiation DNA replication. N-terminal is a conservative zinc finger domain, the center is a catalytic domain with RNA polymerase activity, 266-352 amino acids are TOPRIM domain, this domain is all conservative in topoisomerase and primase, is mainly used for identifying the special sequence of DNA knot domain. This section of sequence is very conservative from virus, thalline, prokaryote to eukaryote. The present embodiment is according to the Mycoplasma ovipneumoniae DnaG gene sequence (accession number is CP118522.1) downloaded by NCBI, different primers and probes (shown in Table 2) are designed, with contrast verification primer and probe of the present invention have specificity.

[0050] The primer sequences for constructing the standard plasmid PUC57-DnaG are:

[0051] DnaG-F: 5'-GTCCAACAAATGTGTTTCTTCCA-3'(SEQ ID NO.1)

[0052] DnaG-R: 5'-TGACGTTGAATACCTTGTTGAAA-3' (SEQ ID NO.2)

[0053] The sequence of the standard plasmid PUC57-DnaG is shown in SEQ ID NO.3:

[0054]

[0055]

[0056] Table 2 Primer information

[0057]

[0058] Reaction system: Premix Ex Taq 12.5μL, upstream and downstream primers 0.5μL each, probe 0.25μL, DNA template 2μL, add deionized water to the total reaction system of 25μL. Reaction program: 95℃2min; 95℃10s, 60℃30s, 72℃20s, 40 cycles.

[0059] Table 3 CT values ​​of primer-probe combinations

[0060]

[0061] The above primers and probes were combined and the CT values ​​of each combination were compared. The results are shown in Table 3 and Figures 1-2As shown. The first combination detected the highest fluorescence intensity and the lowest CT value, which was 12.79, indicating that its specificity and sensitivity had obvious advantages; the CT values ​​of the second and fourth combinations were both less than 20, with little difference from the first combination, and both were better primer sets. Based on the CT value detection results of each combination, when the primer pairs were the same, the CT value of the probe 1 combination was lower than that of the probe 2 combination; and when the probes were the same, the CT values ​​of the F1-R1 and F2-R2 primer pairs were significantly lower than those of the F3-R3 pair.

[0062] Example 2 Optimal Tm value screening

[0063] This example is to screen the optimal annealing temperature, select the optimal primer set (first combination) in Example 1, and select the Tm value with a lower CT value from the range of 53°C to 65°C. The reaction system is as follows: Premix Ex Taq 12.5μL, upstream and downstream primers 0.5μL each, probe 0.25μL, DNA template 2μL, deionized water is added to the total reaction system of 25μL. The reaction procedure is as follows: 95℃2min; 95℃10s, Tm (53℃~65℃)30s, 72℃20s, 40 cycles.

[0064] It was found during the experiment that when the cycle number was 35-45, it could be used for the detection of Mycoplasma ovipneumoniae in the present invention, and when the cycle number was 40, the amplification reached a plateau, so 40 cycles were preferred in the present invention.

[0065] Table 4 CT values ​​corresponding to each Tm value

[0066]

[0067] It can be seen from Table 4 that the lowest CT value is 16.97, and its corresponding Tm value is 57.2°C, so it can be determined that the optimal Tm value is 57.2°C. The difference between the Tm value of 54.8-60.2°C and the CT value at the optimal Tm value is small, and it can also be used for the identification of Mycoplasma ovipneumoniae.

[0068] Example 3 Screening of the optimal amount of primers added

[0069] In this embodiment, the concentrations of the upstream primer solution and the downstream primer solution are both 10 μmol / L. To screen the optimal amount of primers added, the optimal primer-probe combination (first combination) in Example 1 is selected, and a combination with a lower CT value is selected from the volume range of 0.1-1.5 μL. Reaction system: Premix Ex Taq 12.5 μL, primer 0.1-1.5 μL, probe 0.25 μL, DNA template 2 μL, deionized water is added to the total reaction system of 25 μL. Reaction procedure: 95°C pre-denaturation for 2 min; 95°C denaturation for 10 s, 57°C annealing for 30 s, 72°C extension for 20 s, 40 cycles.

[0070] Table 5 The amount of primers added and their corresponding CT values

[0071]

[0072] The contents of the upstream primer and the downstream primer and their corresponding CT values ​​are shown in Table 5. It can be seen that the lowest CT value is 16.85, and the corresponding primer addition amount is 0.7μL / each (i.e. 0.28μM). Therefore, the optimal addition amounts of the upstream primer and the downstream primer in the PCR reaction system are 0.7μL / each (i.e. 0.28μM). The difference between the primer addition amount of 0.3-1.1μL / each (i.e. 0.12-0.44μM) and the optimal addition amount is small, and it can also be used to detect Mycoplasma ovipneumoniae.

[0073] Example 4 Screening of the Optimal Addition Amount of Probe

[0074] The concentration of the probe solution in this embodiment is 10 μmol / L. In order to screen the optimal amount of probe added, the optimal primer-probe combination (first combination) in Example 1 is selected, and a combination with a lower CT value is selected from the volume range of 0.05-0.65 μL. Reaction system: Premix Ex Taq 12.5 μL, 0.7 μL of upstream and downstream primers each, 0.05-0.65 μL of probe, 2 μL of DNA template, and deionized water is added to the total reaction system of 25 μL. Reaction procedure: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 57°C for 30 s, extension at 72°C for 20 s, and 40 cycles.

[0075] Table 6 Probe addition amount and its corresponding CT value

[0076]

[0077]

[0078] The contents of the probes and their corresponding CT values ​​are shown in Table 6. It can be seen that the lowest CT value is 17.12, and the corresponding probe addition amount is 0.25 μL (i.e. 0.10 μM). Therefore, the optimal addition amount of the probe in the qPCR reaction system is 0.25 μL (i.e. 0.10 μM). The difference between the probe addition amount of 0.05-0.45 μL (i.e. 0.02-0.18 μM) and the optimal addition amount is small, and it can also be used to detect Mycoplasma ovipneumoniae.

[0079] Example 5 Linear Range Verification

[0080] The optimal primer-probe combination and amplification conditions optimized in Examples 1-4 were used to perform amplification using eight groups of concentration gradients of the standard substance PUC57-DnaG diluted 10 times in series, and repeated 4 times to verify whether the linear relationship between the amplification concentration and the CT value and the amplification efficiency met the requirements. The results are shown in Table 7, and the amplification curve is shown in Figure 3 shown.

[0081] Table 7 Linear relationship verification results

[0082]

[0083] Through Table 7 and Figure 3 It can be seen that the standard curve R of each group of standards 2 All of them were greater than 0.99, indicating that the linear relationship of each group met the requirements. At the same time, the amplification efficiency E value of each group reached more than 95%, indicating that the amplification efficiency also met the requirements.

[0084] Example 6 Minimum Detection Limit Verification

[0085] Use plasmid dilution to add 2.43 × 10 2 The copies / μL standard was sequentially diluted 10 times in a gradient, and different primer probe combinations and amplification conditions were used. Each concentration was repeated 20 times, and the positive test results for more than 19 times were defined as the minimum detection limit of the method. Therefore, based on Example 5, this example further set the concentrations of 243 copies / μL, 24.3 copies / μL, and 2.43 copies / μL to further determine the specific detection limit. The specific conditions and test results are shown in Table 8.

[0086] Table 8 Minimum detection limit verification results

[0087]

[0088] The minimum detection limit results of the primer sets of the first combination, the second combination, the third combination and the fourth combination are shown in Table 8, wherein the primer sets of the first combination can detect CT values ​​at 243-2.43 copies / μL, which shows that the minimum detection limit of the primer sets of the first combination is 2.43 copies / μL; the primer sets of the second and fourth combinations can detect CT values ​​at 243-24.3 copies / μL, and 15 sets cannot detect CT values ​​at 2.43 copies / μL, which shows that the minimum detection limit of the primer sets of the second and fourth combinations is 24.3 copies / μL.

[0089] Example 7 Repeatability Verification

[0090] In this example, in order to verify the repeatability and stability of the detection method of the present invention, a standard sample (2.43×10 9 -2.43×10 3 copies / μL), different primer probe combinations and optimal amplification conditions were used to perform repeated detection within and between groups, and the experimental data were analyzed to calculate the coefficient of variation. Coefficient of variation (p) = standard deviation (SD) / mean (χ), verifying the stability of this method. The results of the first combination of primer sets, the second combination of primer sets, and the fourth combination of primer sets are shown in Table 9. The CV values ​​within and between groups of each standard are less than 5%, indicating that the repeatability and stability of this method are good.

[0091] Table 9 Repeatability verification results

[0092]

[0093] Example 8 Specificity Verification

[0094] Different primer probe combinations and optimal amplification conditions were used to perform specific verification on a variety of common pathogens. The pathogens used were: Mycoplasma capricolum 87001, Mycoplasma capricolum subsp. capricolum, Mycoplasma mycoides subsp. capricolum PG3, Mycoplasma agalactiae PG2, Mycoplasma bovis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Escherichia coli, Pasteurella mitis, Mannheimia sp., Haemophilus parasuis HPS, and Staphylococcus aureus. The test results are shown in Table 10.

[0095] Table 10 Pathogen solutions and their CT values

[0096]

[0097]

[0098] pass Figure 4 It can be seen from the amplification curves and Table 10 that, using the detection method of the present invention, the three combinations of primer sets have obvious fluorescence amplification signals only when Mycoplasma ovipneumoniae is detected, and the detection result is positive; no CT value is detected for other pathogens, indicating that the three primer sets screened by the present invention have good specificity for detecting Mycoplasma ovipneumoniae.

[0099] Example 9 Fluorescence quantitative PCR and live bacteria linearity verification

[0100] 9.1 Linear establishment

[0101] Take the fermentation liquid of Mycoplasma ovipneumoniae harvested at different times, set 3 parallel samples for each sample to be tested, extract the genome. And use the 10-fold gradient dilution of the standard plasmid as a template, perform fluorescence quantitative PCR detection and draw a standard curve. According to the established standard curve, calculate the copy number of the sample to be tested; take the bacterial liquid of each sample to be tested for CFU method live bacteria count, and calculate the live bacteria count of the sample to be tested according to the dilution and plate live bacteria.

[0102] Table 11 Results of viable bacterial count and fluorescence quantitative PCR detection of Mycoplasma ovipneumoniae

[0103]

[0104] The results of fluorescence quantitative PCR detection of Mycoplasma ovipneumoniae fermentation broth harvested at different times are shown in Table 11. Through the correlation analysis of the results of Mycoplasma ovipneumoniae CFU method detection and the results of fluorescence quantitative PCR method detection, it was found that the two had a very significant linear correlation (R 2 =0.9785), and the established regression equation was lgCFU=0.8032×lgcopy+1.0377.

[0105] 9.2 Linear Verification

[0106] According to step 9.1 of Example 9, 15 portions of Mycoplasma ovipneumoniae fermentation broth harvested at 5 different times (16, 20, and 24 hours) were subjected to fluorescent quantitative PCR detection and viable bacteria count to verify the linear relationship in the fermentation broth samples.

[0107] Table 12 Repeated verification of linear relationship

[0108]

[0109] The viable counts of Mycoplasma ovipneumoniae fermentation cultures harvested from different batches and the results of fluorescence quantitative PCR are shown in Table 12. It can be seen that the theoretical value of lgCFU / mL is not significantly different from the actual value (P>0.05). This shows that the qPCR rapid detection method established by the present invention can quantify the number of viable bacteria in the sample through a linear regression equation with the number of viable bacteria, and can be used for rapid quantitative detection of viable bacteria in fermentation broth containing Mycoplasma ovipneumoniae and vaccine semi-finished products.

[0110] 9.3 Verification of detection rate of clinical samples

[0111] The method established by the present invention, conventional PCR (i.e., amplification using the same primers as those of the present invention, followed by gel electrophoresis to detect the amplified product) and separation and culture methods were used to isolate 113 samples of

[0112] The test results of different types of clinical samples were compared, as shown in Table 13 and Figure 5 shown.

[0113] Table 13 Results of conventional PCR and real-time fluorescence quantitative PCR detection of clinical samples

[0114]

[0115]

[0116] Through Table 13 and Figure 5 It can be seen that the positive rate detected by the method of the present invention is 73.45% (83 / 113), the positive rate detected by the common PCR method is 51.33% (58 / 113), and the samples with positive results based on the common PCR detection results are also positive when tested by fluorescent quantitative PCR, with a coincidence rate of 100%. Based on the samples with negative results based on the common PCR detection results, 25 positive samples were found by fluorescent quantitative PCR detection. It shows that fluorescent quantitative PCR can significantly improve the detection rate of Mycoplasma ovipneumoniae.

[0117] 69 lung tissues and 14 lung tissue samples with positive results of the fluorescent quantitative PCR test of the present invention were separated and identified. As a result, 3 strains of Mycoplasma ovipneumoniae were separated from the lung tissues (the positive rate was 3.61% (3 / 83)). The isolated and purified strains were subjected to gene amplification (see Figure 6 As shown), cloning and sequencing, the results all showed the target gene sequence of Mycoplasma ovipneumoniae, that is, Mycoplasma ovipneumoniae was isolated.

[0118] Example 10 Fluorescence quantitative PCR and total protein linearity verification

[0119] 10.1 Linear Establishment

[0120] Take the fermentation broth of Mycoplasma ovipneumoniae harvested at different times, and set 3 parallel samples for each sample to be tested. Calculate the copy number of the sample to be tested by fluorescent quantitative PCR according to step 9.1 in Example 9; take 1 ml of each sample to be tested, centrifuge at 12000 r / min for 30 min, discard the supernatant, draw an equal volume of buffer and blow the precipitate to suspend and mix, centrifuge at 12000 r / min for 30 min, wash twice in the same way, discard the supernatant after centrifugation, suspend the precipitate with 1 ml of buffer, and finally determine the protein concentration of the corresponding sample using a BCA protein quantitative analysis kit.

[0121] Table 14 Results of total protein and fluorescence quantitative PCR detection of Mycoplasma ovipneumoniae

[0122]

[0123] The total protein content of Mycoplasma ovipneumoniae cultures harvested at different times and the results of fluorescence quantitative PCR are shown in Table 14. The correlation analysis between the total protein content of Mycoplasma ovipneumoniae and the results of fluorescence quantitative PCR showed that the two had a very significant linear correlation (R 2 =0.9758), and the regression equation established was total protein amount = 70.832 × lgcopy - 549.74.

[0124] 10.2 Linear Validation

[0125] According to step 10.1 of Example 10, 15 portions of Mycoplasma ovipneumoniae fermentation broth harvested at 5 different times (16, 20, and 24 hours) were subjected to fluorescence quantitative PCR and total protein detection to verify the linear relationship in the fermentation broth samples.

[0126] Table 15 Repeated verification of linear relationship

[0127]

[0128] The total protein content of Mycoplasma ovipneumoniae cultures harvested from different batches and the results of fluorescence quantitative PCR are shown in Table 15. It can be seen that there is no significant difference between the theoretical value and the actual value of the total protein content (P>0.05). Figure 7 As shown in the data, the number of viable cells and the amount of total protein were also linearly correlated (R 2 =0.9516, and the regression equation is total protein amount = 80.866×lgCFU-591.39). This shows that the established fluorescence quantitative PCR method can be used for rapid quantitative detection of total protein amount in Mycoplasma ovipneumoniae fermentation broth and vaccine semi-finished products.

[0129] In summary, the method established by the present invention can realize the qualitative and quantitative detection of Mycoplasma ovipneumoniae, and effectively solves the problems that the existing quantitative bacterial concentration methods (CCU method and CFU method) of Mycoplasma ovipneumoniae have long detection cycles, cumbersome detection steps, and only rely on visual observation when judging the results, without sufficient reliable basis, which affects the accuracy of the experiment.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection primer set for detecting Mycoplasma ovipneumoniae, characterized in that: The primer set includes an upstream primer and a downstream primer, and the sequence of the upstream primer is shown in SEQ ID NO.4 or SEQ ID NO.6; The downstream primer sequence is shown as SEQ ID NO.5 or SEQ ID NO.

7.

2. The detection primer set according to claim 1, characterized in that: The primer set also includes a probe, and the probe sequence is shown as SEQ ID NO.10 or SEQ ID NO.

11.

3. The detection primer set according to claim 1, characterized in that The primer set is an upstream primer with a sequence as shown in SEQ ID NO.4, a downstream primer with a sequence as shown in SEQ ID NO.5 and a probe with a sequence as shown in SEQ ID NO.

10.

4. The detection primer set according to claim 2, characterized in that: The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; preferably, the fluorescent reporter group is a FAM group, a CY5 group or a HEX group, and the fluorescent quencher group is a MGB group, a BHQ1 group or a BHQ2 group; more preferably, the fluorescent reporter group is a FAM group, and the fluorescent quencher group is a MGB group.

5. A method for detecting Mycoplasma ovipneumoniae, characterized in that: A qPCR reaction system comprising the primer set according to any one of claims 1 to 4 is used to amplify and detect a sample containing Mycoplasma ovipneumoniae.

6. The detection method according to claim 5, characterized in that: The reaction procedure for amplification using the qPCR reaction system is pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s; Anneal at 54.8-60.2℃ for 10s; elongate at 72℃ for 20s, cycle number 35-45; Preferably, the reaction procedure for amplification in the qPCR reaction system is pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s; Anneal at 57.2°C for 10 s; extend at 72°C for 20 s, for 40 cycles.

7. The detection method according to claim 5, characterized in that: The working concentration of the upstream primer is 0.12-0.44 μM, preferably 0.28 μM; The working concentration of the downstream primer is 0.12-0.44 μM, preferably 0.28 μM; The working concentration of the probe is 0.02-0.18 μM, preferably 0.10 μM.

8. A fluorescent quantitative PCR kit comprising the primer set for detecting Mycoplasma ovipneumoniae according to any one of claims 1 to 3.

9. The kit according to claim 8, characterized in that: The kit also includes a positive control substance and / or a negative control substance; preferably, the positive control substance is a recombinant plasmid containing a Mycoplasma ovipneumoniae gene, and the negative control substance is deionized water.

10. Use of the kit according to claim 8 or 9 in the qualitative or quantitative detection of Mycoplasma ovipneumoniae.

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