CN113308561B - Primer group for detecting mycoplasma hyopneumoniae, application thereof and real-time fluorescent quantitative PCR detection method - Google Patents
Primer group for detecting mycoplasma hyopneumoniae, application thereof and real-time fluorescent quantitative PCR detection method Download PDFInfo
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Abstract
The invention provides a primer group for detecting mycoplasma hyopneumoniae, application thereof and a real-time fluorescent quantitative PCR detection method, and relates to the technical field of biology. The primer group for detecting the mycoplasma hyopneumoniae, provided by the invention, comprises the first primer pair and the probe, has the characteristics of strong specificity and high sensitivity, and can be used for detecting the mycoplasma hyopneumoniae. The invention provides a real-time fluorescent quantitative PCR detection method of mycoplasma hyopneumoniae, which adopts the primer group, firstly carries out real-time fluorescent quantitative PCR on a mycoplasma hyopneumoniae standard substance with known initial copy number, and obtains the relation between the copy number and the Ct value; the detection method has the advantages of high specificity, good repeatability, high sensitivity, quick detection and accurate quantification, and has great significance for quick quantitative detection of mycoplasma hyopneumoniae cultures, semi-finished vaccine products and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting mycoplasma hyopneumoniae, application thereof and a real-time fluorescent quantitative PCR detection method.
Background
Mycoplasma hyopneumoniae (Swine enzootic hyopneumoniae), commonly known as mycoplasmosis, is a chronic, contact, respiratory infectious disease caused by Mycoplasma hyopneumoniae. The main clinical symptoms are cough and asthma, and the secondary infection of other germs (such as pasteurella multocida, haemophilus parasuis, actinobacillus pleuropneumoniae and the like) is frequent. The disease is widely distributed around the world and occurs in pig farms in many areas of our country. Due to the existence of the pigs with the bacteria, the production performance of the pig herd is obviously reduced, the daily gain of the infected pig herd is reduced, the feed reward is reduced, the time for marketing is delayed, the growth uniformity of the pig herd is poor, and the cost of medication is increased, so that the relatively serious economic loss is caused to the pig farm.
Since Mycoplasma hyopneumoniae is small and yields are low, the commonly used methods for quantitative assessment of bacterial cells (such as turbidity, cell volume and dry weight) are not suitable for quantitative detection of Mycoplasma hyopneumoniae. The existing mycoplasma hyopneumoniae quantitative method is mainly a CCU detection method, the method is time-consuming and labor-consuming, identification and counting errors are often caused, pollution is easy to occur in the experimental process, and the result has volatility.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a primer group for detecting mycoplasma hyopneumoniae, which has strong specificity and can realize the detection of mycoplasma hyopneumoniae.
The second purpose of the invention is to provide an application of the primer group in the detection of the mycoplasma hyopneumoniae.
The third objective of the present invention is to provide a real-time fluorescent quantitative PCR detection method for Mycoplasma hyopneumoniae, which solves at least one of the above problems.
In a first aspect, the invention provides a primer set for detecting mycoplasma hyopneumoniae, the primer set comprising a first primer pair and a probe;
the first primer pair has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the probe comprises a nucleotide sequence shown as SEQ ID NO. 3.
As a further technical scheme, the 5 'end and the 3' end of the probe respectively contain a fluorescence reporter group and a fluorescence quenching group.
As a further technical solution, the fluorescent reporter group comprises FAM, CY5 or HEX, preferably FAM.
As a further technical scheme, the fluorescence quenching group comprises TAMRA, BHQ1 or BHQ2, preferably TAMRA.
In a second aspect, the invention provides an application of a primer group in mycoplasma hyopneumoniae detection.
In a third aspect, the invention provides a real-time fluorescent quantitative PCR detection method for mycoplasma hyopneumoniae, which comprises the following steps: carrying out real-time fluorescent quantitative PCR on the mycoplasma hyopneumoniae standard substance with known initial copy number to obtain the relation between the copy number and the Ct value; extracting DNA of a sample to be detected, carrying out real-time fluorescence quantitative PCR, and calculating the copy number of a target gene in the sample to be detected;
the real-time fluorescent quantitative PCR adopts the primer group.
As a further technical scheme, the mycoplasma hyopneumoniae standard comprises a plasmid.
As a further technical solution, the plasmid comprises a sequence of mycoplasma hyopneumoniae amplified by the first primer pair;
preferably, the plasmid comprises a sequence of Mycoplasma hyopneumoniae amplified by the second primer pair;
the second primer pair has nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO. 5.
As a further technical scheme, the concentration of a first primer pair in a reaction system of the real-time fluorescent quantitative PCR is 0.1-1.0 mu mol/L, and the concentration of a probe is 0.1-1.0 mu mol/L;
preferably, the concentration of the first primer pair in the reaction system of the real-time fluorescent quantitative PCR is 0.4 mu mol/L, and the concentration of the probe in the reaction system of the real-time fluorescent quantitative PCR is 0.3 mu mol/L.
As a further technical scheme, the reaction conditions of the real-time fluorescence quantitative PCR are as follows: pre-denaturation: 93-95 deg.C, 1-3min, 1 cycle; denaturation: 93-95 ℃ for 8-15s, annealing: at 55-65 deg.C for 25-35s for 40 cycles;
preferably, the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denaturation: at 94 ℃, 2min, 1 cycle; denaturation: 94 ℃, 10s, annealing: 60 ℃, 30s, 40 cycles.
Compared with the prior art, the invention has the following beneficial effects:
the primer group for detecting the mycoplasma hyopneumoniae, provided by the invention, comprises the first primer pair and the probe, has the characteristics of strong specificity and high sensitivity, and can be used for detecting the mycoplasma hyopneumoniae.
The invention provides a real-time fluorescent quantitative PCR detection method of mycoplasma hyopneumoniae, which adopts the primer group, firstly carries out real-time fluorescent quantitative PCR on a mycoplasma hyopneumoniae standard substance with known initial copy number, and obtains the relation between the copy number and the Ct value; and then extracting the DNA of the sample to be detected, performing real-time fluorescence quantitative PCR, and calculating to obtain the copy number of the target gene in the sample to be detected.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram showing the PCR amplification result of Mycoplasma hyopneumoniae genome provided by the invention; m: marker; -: negative control; mhp: mycoplasma hyopneumoniae bacterial liquid;
FIG. 2 is a schematic diagram of the restriction enzyme identification result of the Mycoplasma hyopneumoniae recombinant plasmid provided by the invention; m: marker; -: negative control; mhp: a mycoplasma hyopneumoniae recombinant plasmid;
FIG. 3 is a schematic diagram showing the PCR identification result of the Mycoplasma hyopneumoniae recombinant plasmid provided by the invention; m: marker; -: negative control; mhp: mycoplasma hyopneumoniae recombinant plasmids;
FIG. 4 is a schematic diagram of an amplification curve of a fluorescent quantitative PCR standard plasmid for Mycoplasma hyopneumoniae provided by the invention;
FIG. 5 is a schematic diagram of a standard curve of a fluorescent quantitative PCR standard plasmid for Mycoplasma hyopneumoniae provided by the invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the specific conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides a primer set for detecting mycoplasma hyopneumoniae, the primer set comprising a first primer pair and a probe;
the first primer pair has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the probe comprises a nucleotide sequence shown as SEQ ID NO. 3.
The first primer pair and the probe for detecting the mycoplasma hyopneumoniae provided by the invention are designed by taking a highly conserved region in a 16S rRNA gene genus which has specificity and is a main target gene for bacteria classification identification as an amplification region, have the characteristics of strong specificity and high sensitivity, and can be used for detecting the mycoplasma hyopneumoniae.
In some preferred embodiments, the 5 'end and the 3' end of the probe contain a fluorescent reporter group and a fluorescent quencher group, respectively. In this embodiment, the type of the fluorescence reporter group and the fluorescence quencher group contained in the probe is not particularly limited, and any group that can be linked to the probe and can perform a corresponding characterization function may be used.
In some preferred embodiments, the fluorescent reporter comprises FAM, CY5, or HEX, preferably FAM.
In some preferred embodiments, the fluorescence quenching group comprises TAMRA, BHQ1, or BHQ2, preferably TAMRA.
In a second aspect, the invention provides an application of a primer group in mycoplasma hyopneumoniae detection.
The primer group for detecting the mycoplasma hyopneumoniae provided by the invention has the characteristics of strong specificity and high sensitivity, and can be used for detecting the mycoplasma hyopneumoniae.
In a third aspect, the invention provides a real-time fluorescent quantitative PCR detection method for Mycoplasma hyopneumoniae, which comprises the following steps: carrying out real-time fluorescent quantitative PCR on the mycoplasma hyopneumoniae standard substance with known initial copy number to obtain the relation between the copy number and the Ct value; extracting DNA of a sample to be detected, carrying out real-time fluorescence quantitative PCR, and calculating the copy number of a target gene in the sample to be detected;
the real-time fluorescent quantitative PCR adopts the primer group.
The real-time fluorescent quantitative PCR detection method for the mycoplasma hyopneumoniae provided by the invention has the advantages of high specificity, good repeatability, high sensitivity, rapid detection and accurate quantification, and has great significance for rapid quantitative detection of mycoplasma hyopneumoniae cultures, vaccine semi-finished products and the like.
In some preferred embodiments, the mycoplasma hyopneumoniae standard includes, but is not limited to, a plasmid.
In some preferred embodiments, the plasmid comprises a sequence of Mycoplasma hyopneumoniae amplified by the first primer pair, capable of being amplified by the first primer set.
Preferably, the plasmid comprises a sequence of Mycoplasma hyopneumoniae amplified by the second primer pair. In the mycoplasma hyopneumoniae genome, the amplification product of the first primer pair is located within the sequence amplified by the second primer pair and can also be amplified by the first primer set.
The second primer pair has nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO. 5.
In some preferred embodiments, the concentration of the first primer pair in the reaction system of the real-time fluorescent quantitative PCR is 0.1-1.0 μmol/L, and the concentration of the probe is 0.1-1.0 μmol/L;
preferably, the concentration of the first primer pair in the reaction system of the real-time fluorescent quantitative PCR is 0.4 mu mol/L, and the concentration of the probe is 0.3 mu mol/L.
In the invention, the concentration of the first primer pair and the probe in the reaction system of the real-time fluorescence quantitative PCR is further optimized and adjusted, thereby being more beneficial to the PCR reaction.
In some preferred embodiments, the reaction conditions of the real-time fluorescent quantitative PCR are: pre-denaturation: 93-95 deg.C, 1-3min, 1 cycle; denaturation: 93-95 ℃, 8-15s, annealing: at 55-65 deg.C for 25-35s for 40 cycles;
preferably, the reaction conditions of the real-time fluorescent quantitative PCR are as follows: pre-denaturation: 94 ℃, 2min, 1 cycle; denaturation: 94 ℃, 10s, annealing: 60 ℃, 30s, 40 cycles.
By further optimizing and adjusting the reaction conditions of the real-time fluorescence quantitative PCR, the method is beneficial to the PCR reaction and shortens the detection period.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
1 primer and Probe design and Synthesis
Aiming at a highly conserved region in a 16S rRNA gene genus which has specificity and is a main target gene for bacteria classification identification as an amplification region, a first primer pair realtime F/R of Real Time PCR and a second primer pair clone F/R for constructing a standard plasmid are designed, wherein the sequences of the primers and the probe are as follows:
realtime F：5’-AACTGGTCATATATTGACACTAAGGG-3’(SEQ ID NO.1)；
realtime R：5’-TGTGTTAGTGACTTTTGCCACCAACT-3’(SEQ ID NO.2)。
and (3) probe: 5'-CAGGATTAGATACCCTGGTAGTCCAC-3' (SEQ ID NO. 3);
clone F：5’-GAGCCTTCAAGCTTCACCAAGA-3’(SEQ ID NO.4)；
clone R：5’-GCGGATCATTTAACGCGTTAGC-3’(SEQ ID NO.5)。
the product of primer pair realtime F/R is located within the amplified sequence of primer pair clone F/R. The 5 'end of the Real Time PCR probe is combined with a fluorescence reporter group (FAM), and the 3' end is combined with a fluorescence quenching group (TAMRA).
The above primers and probes were synthesized by Shanghai bioengineering Inc.
2 culture of Mycoplasma hyopneumoniae
Inoculating 1.5mL of Mycoplasma hyopneumoniae CJ strain to 10mL of improved Fris culture medium, culturing in a constant-temperature incubator at the temperature of 36-38 ℃ for 48h, subculturing according to the ratio of 1: 10(V/V), culturing in the constant-temperature incubator at the temperature of 36-38 ℃, harvesting a bacterial solution when the color of the culture medium turns yellow and the pH value is reduced from 7.6 to 6.7-6.8, and preserving at-20 ℃.
3DNA extraction
The harvested bacterial liquid is subjected to nucleic acid extraction according to the instruction of a Takara bacterial genome DNA extraction kit.
4DNA Standard preparation
The genome extracted from the collected mycoplasma hyopneumoniae bacterial liquid is amplified with clone F and clone R as primers to obtain mycoplasma hyopneumoniae 16S rRNA gene, and the PCR product is electrophoretically separated to obtain specific band of about 670bp, which is identical to the size of the target segment (see FIG. 1). Then gel cutting, purifying, quantifying, determining the concentration of target gene, cloning into pMD19-T vector, transforming DH5 alpha competent cell, enzyme digestion identification (as figure 2) and PCR identification (as figure 3) are positive colony, after proliferation culture, extracting plasmid and sending into Shanghai bioengineering technology service company LimitedAnd (5) performing quantification and sequencing. The positive plasmid with correct sequence is analyzed by ultramicro nucleic acid and protein analyzer to determine plasmid concentration and purity, according to the molecular mass and mass concentration of recombinant plasmid the plasmid copy number per unit volume is calculated, and through serial dilution the recombinant plasmid is diluted to 101～109The samples/. mu.L are used as standard substance for standby.
5 optimization of the reaction System
In a reaction system of a template with the same concentration, using a realtime F and a realtime R as primers, and optimizing the concentration of the primers (0.1-1.0 mu mol/L) and the concentration of probes (0.1-1.0 mu mol/L) by a matrix method. Meanwhile, in the TaqMan fluorescent quantitative PCR reaction condition, the optimal annealing temperature (50-68 ℃) is optimized by detecting the positive nucleic acid template with the same concentration. The optimization result is judged by comparing Ct values, the primer concentration is determined to be 0.4 mu mol/L, the probe concentration is determined to be 0.3 mu mol/L, the cycle parameters are pre-denaturation at 94 ℃ for 2 minutes, denaturation at 94 ℃ for 10 seconds, annealing/extension at 60 ℃ for 30 seconds, and the annealing/extension process is repeated for 40 cycles. The results are shown in Table 1.
TABLE 1 results of orthogonal experiments and differential analysis
Drawing of 6 standard curves
Positive plasmids were serially diluted 10-fold as standard (10)3～109copies/. mu.L) as a template, amplified by a Bio-Rad IQ5 real-time fluorescent quantitative PCR instrument, repeated 2 times for each dilution, and analyzed by Bio-Rad IQ5 real-time fluorescent quantitative PCR analysis software to draw a standard curve, and the results are shown in FIG. 4 and FIG. 5.
7 specificity test of real-time fluorescent quantitative PCR
The specificity test is carried out by using nucleic acids of mycoplasma hyopneumoniae, mycoplasma hyorhinis, porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine pseudorabies virus, porcine pasteurella multocida, porcine parvovirus, porcine circovirus type 2 virus, escherichia coli DH5 alpha and BL21 strain as templates and using an optimized real-time fluorescent quantitative PCR condition. The results show that the method has good specificity. The results are shown in Table 2.
TABLE 2 results of specificity test
8 real-time fluorescent quantitative PCR sensitivity test
Mycoplasma hyopneumoniae positive plasmid (10) serially diluted 10-fold0～1010copies/. mu.L) as template, and sensitivity test was performed by Bio-Rad IQ5 real-time fluorescent quantitative PCR analysis software. The results show that the method has good sensitivity. The results are shown in Table 3.
TABLE 3 results of sensitivity test
9 repeatability test of real-time fluorescent quantitative PCR
And 8 parts of nucleic acid samples are taken as templates for detection, each sample is repeatedly detected for 5 times, the Ct15 value is counted, and the variation coefficient is calculated. The results show that the method has good repeatability. The results are shown in Table 4.
TABLE 4 results of the repeatability tests
Example 2
Comparison of the results of detection of Mycoplasma hyopneumoniae Using the CCU method and the fluorescent quantitative PCR method
1 culture of Mycoplasma hyopneumoniae
And (3) taking the birth seeds from the strain library, dissolving the birth seeds by using an improved Fris liquid culture medium, subculturing the birth seeds in the improved Fris liquid culture medium according to a ratio of 1: 10, putting the birth seeds into a constant-temperature incubator at the temperature of 36-38 ℃ for culturing for 48-72 hours, collecting the strain liquid when the color of the strain liquid is changed and the pH value is reduced to 6.70-6.80, taking the strain liquid as a primary seed liquid, and carrying out pure inspection. The second-level seed and the third-level seed are propagated and identified according to the method; adding the third-level seed liquid into the improved Fris liquid culture medium according to the ratio of 1: 10, sealing the opening of a fermentation tank, introducing sterile air, closing all inlets and outlets on the fermentation tank when the pressure in the fermentation tank reaches 0.08MPa, maintaining the pressure, culturing at the temperature of 36-38 ℃, taking out the culture in sterile conditions for 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90 and 96 hours respectively, carrying out pure inspection and determination of the bacterial content, and storing at the temperature of-20 ℃.
2 measurement of the bacterial content of Mycoplasma hyopneumoniae by the CCU method
Sequentially diluting Mycoplasma hyopneumoniae bacterial liquid obtained at different times by 10 times in series until the dilution reaches 10-1、10-2、10-3……10-12And then setting a culture medium as a reference, culturing for 14 days in a constant-temperature incubator at the temperature of 36-38 ℃, observing and recording the color change and the turbidity change of the culture medium every day until the pH value of the culture is not changed, and finally, determining the dilution with the color change as the CCU titer of the culture and determining for 3 times. The results of the CCU assay of M.hyopneumoniae cultures harvested at different times are shown in Table 5.
3 using fluorescent quantitative PCR method to determine the content of mycoplasma hyopneumoniae
And (3) extracting the genome by taking the mycoplasma hyopneumoniae bacterial solutions harvested at different times, wherein each sample to be detected is provided with 3 parallel samples according to the method for extracting the genome in the embodiment 1. And performing fluorescence quantitative PCR by using a 10-fold gradient dilution of the standard plasmid as a template, and calculating the copy number of the sample to be detected according to the established standard curve. The results of the fluorescent quantitative PCR method for the Mycoplasma hyopneumoniae cultures harvested at different times are shown in Table 5.
TABLE 5 CCU and fluorescent quantitative PCR results for Mycoplasma hyopneumoniae detection
Comparison of results of 42 kinds of quantitative detection of Mycoplasma hyopneumoniae
The 2 quantitative detection results of the mycoplasma hyopneumoniae culture show that the correlation between the detection result of the CCU method and the detection result of the fluorescent quantitative PCR method is very obvious (R)20.95) and establishing a regression equation of lgCCU of 1.6778 × lgcopy-6.7697.
Application of 5 real-time fluorescent quantitative PCR detection method
18 M. hyopneumoniae cultures harvested at 6 different times (36, 42, 48 hours) were obtained according to example 1, quantitatively assayed for bacterial content according to examples 2 and 3, and analyzed for results (see Table 6). The results show that the theoretical and actual values of lgCCU/mL do not differ significantly (P > 0.05). Therefore, the established fluorescent quantitative PCR method can be used for quickly and quantitatively detecting the mycoplasma hyopneumoniae culture and the vaccine semi-finished product.
TABLE 6 application of method for real-time fluorescent quantitative PCR detection of mycoplasma hyopneumoniae
Comparison of 62 detection methods
The results of 8 samples detected by the research method and the commercial kit (Shanghai Husha Mycoplasma hyopneumoniae nucleic acid detection kit (PCR-fluorescent probe method)) show that the method has lower Ct value and higher detection rate. The results are shown in Table 7.
TABLE 72 comparison of test methods
| Sample numbering | The present research method | |
| Sample | ||
| 1 | 19.10 | 19.37 |
| |
17.20 | 19.41 |
| Sample 3 | 28.65 | 32.81 |
| Sample No.4 | 33.34 | 37.43 |
| Sample No.5 | 17.95 | 19.10 |
| Sample No. 6 | 24.14 | 28.40 |
| Sample 7 | 32.07 | 35.22 |
| |
34.55 | 37.67 |
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Tiankang pharmaceutical (Suzhou) Co., Ltd
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Claims (15)
1. A primer group for detecting mycoplasma hyopneumoniae, which is characterized by comprising a first primer pair and a probe;
the first primer pair has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
the probe comprises a nucleotide sequence shown as SEQ ID NO. 3.
2. The primer set for detecting mycoplasma hyopneumoniae of claim 1, wherein the 5 'end and the 3' end of the probe respectively comprise a fluorescence reporter group and a fluorescence quencher group.
3. The primer set for detecting mycoplasma hyopneumoniae of claim 2, wherein the fluorescent reporter group comprises FAM, CY5, or HEX.
4. The primer set for detecting mycoplasma hyopneumoniae of claim 3, wherein the fluorescent reporter group is FAM.
5. The primer set for detecting mycoplasma hyopneumoniae of claim 2, wherein the fluorescence quenching group comprises TAMRA, BHQ1 or BHQ 2.
6. The primer set for detecting mycoplasma hyopneumoniae of claim 5, wherein the fluorescence quenching group is TAMRA.
7. Use of the primer set according to any one of claims 1 to 6 for the preparation of a mycoplasma hyopneumoniae detection product.
8. Use according to claim 7, characterized in that the method of detection of the product comprises the following steps: carrying out real-time fluorescent quantitative PCR on the mycoplasma hyopneumoniae standard substance with known initial copy number to obtain the relation between the copy number and the Ct value; extracting DNA of a sample to be detected, carrying out real-time fluorescence quantitative PCR, and calculating the copy number of a target gene in the sample to be detected;
the real-time fluorescent quantitative PCR uses the primer set according to any one of claims 1 to 6.
9. The use of claim 8, wherein the mycoplasma hyopneumoniae standard comprises a plasmid.
10. The use of claim 9, wherein the plasmid comprises a sequence of Mycoplasma hyopneumoniae amplified by the first primer pair.
11. The use of claim 10, wherein the plasmid comprises a sequence of Mycoplasma hyopneumoniae amplified by the second primer pair;
the second primer pair has nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO. 5.
12. The use of claim 8, wherein the concentration of the first primer pair in the reaction system of the real-time fluorescent quantitative PCR is 0.1-1.0 μmol/L, and the concentration of the probe is 0.1-1.0 μmol/L.
13. The use of claim 12, wherein the reaction system of the real-time fluorescence quantitative PCR comprises the first primer pair at a concentration of 0.4 μmol/L and the probe at a concentration of 0.3 μmol/L.
14. The use according to any one of claims 8 to 13, wherein the reaction conditions of the real-time fluorescent quantitative PCR are: pre-denaturation: 93-95 deg.C, 1-3min, 1 cycle; denaturation: 93-95 ℃, 8-15s, annealing: at 55-65 deg.c for 25-35 sec for 40 cycles.
15. The use according to claim 14, wherein the reaction conditions of the real-time fluorescent quantitative PCR are: pre-denaturation: 94 ℃, 2min, 1 cycle; denaturation: 94 ℃, 10s, annealing: 60 ℃, 30s, 40 cycles.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492741A (en) * | 2009-01-22 | 2009-07-29 | 中国农业科学院北京畜牧兽医研究所 | Method for quantitative detection of mycoplasma hyopneumoniae |
| CN103571960A (en) * | 2013-11-08 | 2014-02-12 | 广东温氏食品集团股份有限公司 | Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae |
| CN106701942A (en) * | 2016-12-27 | 2017-05-24 | 湖南新南方养殖服务有限公司 | Real-time fluorescence PCR detection reagent kit for mycoplasma hyopneumoniae of pigs and purpose of real-time fluorescence PCR detection reagent kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492741A (en) * | 2009-01-22 | 2009-07-29 | 中国农业科学院北京畜牧兽医研究所 | Method for quantitative detection of mycoplasma hyopneumoniae |
| CN103571960A (en) * | 2013-11-08 | 2014-02-12 | 广东温氏食品集团股份有限公司 | Absolute fluorescent quantitative polymerase chain reaction (PCR) primer pair, probe and method for determining growth titer of mycoplasma hyopneumoniae |
| CN106701942A (en) * | 2016-12-27 | 2017-05-24 | 湖南新南方养殖服务有限公司 | Real-time fluorescence PCR detection reagent kit for mycoplasma hyopneumoniae of pigs and purpose of real-time fluorescence PCR detection reagent kit |
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