阅读说明：本技术 一种同时检测猪肺炎支原体和猪衣原体的巢氏pcr引物组及其应用 (Nested PCR (polymerase chain reaction) primer group for simultaneously detecting mycoplasma hyopneumoniae and chlamydia suis and application thereof ) 是由 刘国平 谭旭 杨杰 万佳佳 闫春梅 曾攀 胡利群 于 2021-09-29 设计创作，主要内容包括：本发明提供了一种同时检测猪肺炎支原体和猪衣原体的巢氏PCR引物组及其应用,属于分子诊断技术领域。本发明提供的同时检测猪肺炎支原体和猪衣原体的巢氏PCR引物组包括两对外侧引物Mhp-O-F、Mhp-O-R和C.suis-O-F、C.suis-O-R和两对内侧引物Mhp-I-F、Mhp-I-R和C.suis-I-F、C.suis-I-R。本发明以Mhp M12916S rDNA基因序列和C.suis ompA基因保守序列作为检测猪肺炎支原体和猪衣原体的诊断靶标,设计了巢氏PCR的两对外侧引物和两对内侧引物,能够同时检测猪肺炎支原体和猪衣原体,两轮PCR提高了PCR的灵敏性、抗干扰性和特异性,克服了普通PCR检测灵敏度低,特异性差等问题,与现有的间接免疫荧光,血清学等检测方法相比成本较低,检测周期短。(The invention provides a nested PCR primer group for simultaneously detecting mycoplasma hyopneumoniae and chlamydia suis and application thereof, belonging to the technical field of molecular diagnosis. The nested PCR primer group for simultaneously detecting the mycoplasma hyopneumoniae and the chlamydia suis provided by the invention comprises two pairs of outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, and two pairs of inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R. The invention takes Mhp M12916S rDNA gene sequence and C.suis ompA gene conserved sequence as diagnosis targets for detecting mycoplasma hyopneumoniae and chlamydia suis, designs two pairs of outer primers and two pairs of inner primers of nested PCR, can simultaneously detect mycoplasma hyopneumoniae and chlamydia suis, improves the sensitivity, anti-interference and specificity of PCR by two rounds of PCR, overcomes the problems of low detection sensitivity, poor specificity and the like of common PCR, and has lower cost and short detection period compared with the existing detection methods of indirect immunofluorescence, serology and the like.)

1. A nested PCR primer group for simultaneously detecting mycoplasma hyopneumoniae and chlamydia suis is characterized by comprising two pairs of outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, and two pairs of inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R;

the nucleotide sequences of Mhp-O-F, Mhp-O-R are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the C.suis-O-F and the C.suis-O-R are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of Mhp-I-F, Mhp-I-R are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the C.suis-I-F and the C.suis-I-R are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.

2. The use of the primer set of claim 1 in the preparation of a product for detecting mycoplasma hyopneumoniae and chlamydia suis.

3. Use according to claim 2, wherein the product comprises a kit, a reagent and/or a test strip.

4. The use according to claim 2, wherein the method for detecting mycoplasma hyopneumoniae and chlamydia hyopneumoniae by using the primer set according to claim 1 comprises the following steps: extracting DNA of a sample to be detected, carrying out nested PCR, carrying out first round PCR amplification by adopting outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, carrying out second round PCR amplification by adopting inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R, and carrying out electrophoresis detection on a PCR product of each time to detect mycoplasma hyopneumoniae and chlamydia hyopneumoniae.

5. The use of claim 4, wherein the sample to be tested is a porcine blood or tissue sample, or a bronchial lavage, a porcine anal swab, or a nasal swab sample.

6. The use of claim 4, wherein the reaction system of the first round of PCR comprises 12.5 parts by volume of Taq enzyme, 1 part by volume of each of the outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, 1 part by volume of DNA template, and 1 part by volume of ddH₂And O2.5 parts.

7. The use of claim 4, wherein the reaction conditions of the first round of PCR comprise: 94 ℃ for 5min, 94 ℃ for 30s, 52-56 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

8. The use of claim 4, wherein the reaction system of the second round of PCR comprises 12.5 parts by volume of Taq enzyme, 1 part by volume of each of the inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R, 1 part by volume of DNA template, and 1 part by volume of ddH₂And O2.5 parts.

9. The use of claim 4, wherein the reaction conditions of the second round of PCR comprise: 94 ℃ for 5min, 94 ℃ for 30s, 54-58 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

10. The use of claim 4, wherein the two-round PCR shows positive Mycoplasma hyopneumoniae if the band appears at 418bp or 275 bp; if a band appears at 562bp or 395bp, the swine chlamydia is indicated to be positive.

Technical Field

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a nested PCR primer group for simultaneously detecting mycoplasma hyopneumoniae and porcine chlamydia and application thereof.

Background

Mycoplasma hyopneumoniae (Mhp) is a pathogenic microorganism that causes Mycoplasma hyopneumoniae (MPS). The mycoplasma pneumonia of swine is widely spread in all pig raising areas all over the world, and the increase of the medicine cost and the reduction of the pig production performance caused by the spread of the disease cause huge loss to the pig raising industry every year. In addition to causing losses directly from mycoplasma hyopneumoniae, mycoplasma hyopneumoniae is also one of the major causes of porcine respiratory syndrome (PRDC).

Porcine Chlamydiosis (c. suis) is a chronic infectious contact disease caused by certain strains of chlamydophila psittaci (old called chlamydia psittaci), also known as epidemic abortion, porcine chlamydia abortion. Clinically, the disease can be manifested by abortion, stillbirth and weak farrowing of pregnant sows, pneumonia, enteritis, pleuritis, pericarditis, arthritis of newborn piglets, orchitis of breeding boars and the like. Different syndrome groups appear due to the virulence of the strain, the sex, age, physiological conditions and environmental changes of pigs. The disease of swine chlamydia was first identified in countries of the world in the 60's of the 20 th century, and then was successively reported in many countries. In China, in the early 80 s of the 20 th century, Chlamydia psittaci was isolated from sheep abortion and swine reproductive disorder mordants, thereby confirming the existence of Chlamydia disease in animals in China.

The two pathogens are common exogenous contamination viruses of the swine vaccines, and after the vaccines are inoculated with the vaccines polluted by mycoplasma hyopneumoniae or chlamydia suis, the vaccines have acute or continuous harm to the health of swinery, and the similar clinical reports occur frequently. The method also reflects the limitation that the detection method for the two exogenous viruses in China has low sensitivity, so that the detection sensitivity of the two pathogens is improved at present.

In recent years, researchers at home and abroad establish the technologies of pathogen isolation culture, giemsa staining, immunofluorescence assay, serology assay, common PCR diagnosis, common PCR detection and the like to detect Mhp and C.suis. Isolation and culture of pathogens is the gold standard for diagnosis of all pathogenic microorganisms, including mycoplasma, but in most cases is not used as a laboratory tool for the definitive diagnosis of mycoplasma hyopneumoniae and chlamydia suis infection; mainly because of the difficulty and slowness inherent in mycoplasma isolation. In the laboratory, the method of immunofluorescence is mostly used for detecting pathogens, but the method has the defect of insufficient sensitivity. Serological diagnostic methods can be applied to detect whether a population is infected with mycoplasma hyopneumoniae and chlamydia suis, but such methods are not applicable to individuals, because the time for the antibodies of the individuals infected with mycoplasma hyopneumoniae and chlamydia suis to turn positive under the influence of various virulence strains, individual health states, maternal antibody levels and other factors varies from several weeks to several months. Polymerase Chain Reaction (PCR) is a rapid feature as one of the currently used laboratory diagnostic methods for detecting mycoplasma hyopneumoniae and chlamydia suis, but it is noted that the detection of mycoplasma hyopneumoniae and chlamydia suis by ordinary PCR is easy to have false positive and cross infection. Therefore, there is a need in the art to provide a method for simultaneously detecting mycoplasma hyopneumoniae and chlamydia hyopneumoniae with high specificity and sensitivity.

Disclosure of Invention

In view of the above, the invention aims to provide a nested PCR primer set capable of detecting mycoplasma hyopneumoniae and chlamydia suis simultaneously and an application thereof, wherein the nested PCR primer set and the detection method have high specificity and sensitivity, and the lowest copy number of a pathogen capable of being detected by the nested PCR primer set and the detection method is 10⁰And (4) respectively.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a nested PCR primer group for simultaneously detecting mycoplasma hyopneumoniae and chlamydia suis, which comprises two pairs of outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, and two pairs of inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R;

the nucleotide sequences of Mhp-O-F, Mhp-O-R are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the C.suis-O-F and the C.suis-O-R are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of Mhp-I-F, Mhp-I-R are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the C.suis-I-F and the C.suis-I-R are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.

The invention also provides application of the primer group in preparation of products for detecting mycoplasma hyopneumoniae and chlamydia suis.

Preferably, the product comprises a kit, a reagent and/or a test strip.

Preferably, the method for detecting mycoplasma hyopneumoniae and chlamydia suis by using the primer set comprises the following steps: extracting DNA of a sample to be detected, carrying out nested PCR, carrying out first round PCR amplification by adopting outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, carrying out second round PCR amplification by adopting inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R, and carrying out electrophoresis detection on a PCR product of each time to detect mycoplasma hyopneumoniae and chlamydia hyopneumoniae.

Preferably, the sample to be detected is a pig blood sample or a tissue sample, or a bronchial lavage fluid, a pig anus swab or a nose swab sample.

Preferably, the reaction system of the first round of PCR comprises 12.5 parts of Taq enzyme by volume parts, 1 part of each of outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, 1 part of DNA template and 1 part of ddH₂And O2.5 parts.

Preferably, the reaction conditions of the first round of PCR include: 94 ℃ for 5min, 94 ℃ for 30s, 52-56 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

Preferably, the reaction system of the second round of PCR comprises 12.5 parts of Taq enzyme by volume parts, 1 part of each of the inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R,DNA template 1 part, ddH₂And O2.5 parts.

Preferably, the reaction conditions of the second round of PCR include: 94 ℃ for 5min, 94 ℃ for 30s, 54-58 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

Preferably, if a band appears at 418bp or 275bp in the two-round PCR, the mycoplasma hyopneumoniae is positive; if a band appears at 562bp or 395bp, the swine chlamydia is indicated to be positive.

The invention has the beneficial effects that:

the invention aims at the characteristics of mycoplasma hyopneumoniae genome and chlamydia suis genome, applies molecular biology technology, develops and develops a dual nested PCR primer group with high specificity, strong sensitivity and high accuracy by using a mycoplasma hyopneumoniae M129 sequence 16S rDNA gene conserved sequence and a mycoplasma hyopneumoniae ompA gene, and a detection method. The method is simple and easy to implement, the sample source is wide, the sample can be blood samples, tissues, semen, lymph nodes, placenta, pig lung samples, pig lung bronchus samples, fetuses, mesenteries, bronchus lavage fluid, serum samples, pig anus swabs, nose swabs, aerosol, environmental samples and the like, the detection rate is high, the pathogen with single copy number can be successfully detected, and the method provides convenience for monitoring large-scale pig farm mycoplasma hyopneumoniae or chlamydia hyoscyami with low toxicity. Meanwhile, the primer group disclosed by the invention can be suitable for early warning detection, clinical diagnosis, immune evaluation and the like.

Drawings

FIG. 1 is a gel electrophoresis diagram of double nested PCR detection of swine chlamydia and mycoplasma, wherein A is an outer primer detection gel electrophoresis diagram, M is a Marker, 1 and 2 are two parallel tests, B is an inner primer detection gel electrophoresis diagram, M is a Marker, and 1 and 2 are two parallel tests;

FIG. 2 is a gel electrophoresis diagram of dual nested PCR primer specificity detection of swine chlamydia and mycoplasma, wherein A is an outer primer result diagram, B is an inner primer result diagram, M in A and B is DL2000 DNAmarker, 1 is a positive control, 2 is hog cholera virus positive, 3 is porcine parvovirus positive, 4 is porcine pseudorabies virus positive, 5 is porcine reproductive and respiratory syndrome virus positive, 6 is Escherichia coli positive, 7 is Staphylococcus aureus positive, and 8 is sterile distilled water negative control;

FIG. 3 is a gel electrophoresis diagram of dual nested PCR primer sensitivity detection of swine chlamydia and mycoplasma, wherein A is the result diagram of the outer primer, B is the result diagram of the inner primer, M in both diagrams A and B is DL2000 DNAmarker, and template copy number is 5 × 10 in sequence in 1-10⁹、5×10⁸、5×10⁷、5×10⁶、5×10⁵、5×10⁴、5×10³、5×10²、5×10¹、5×10⁰；

FIG. 4 is a gel electrophoresis diagram of the anti-interference test of the dual nested PCR primers for swine chlamydia and mycoplasma, wherein A is the result diagram of the outer primer, B is the result diagram of the inner primer, M in both diagrams A and B is DL2000 DNAmarker, 1 is a positive mixture of swine mycoplasma, chlamydia recombinant plasmid and swine fever virus, 2 is a positive mixture of swine mycoplasma, chlamydia recombinant plasmid and swine parvovirus, 3 is a positive mixture of swine mycoplasma, chlamydia recombinant plasmid and swine pseudorabies virus, 4 is a mixture of swine mycoplasma, chlamydia recombinant plasmid and Escherichia coli, and 5 is sterile enzyme-free water.

Detailed Description

The invention provides a nested PCR primer group for simultaneously detecting mycoplasma hyopneumoniae and chlamydia suis, which comprises two pairs of outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, and two pairs of inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R;

the nucleotide sequences of Mhp-O-F, Mhp-O-R are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the C.suis-O-F and the C.suis-O-R are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of Mhp-I-F, Mhp-I-R are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the C.suis-I-F and the C.suis-I-R are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8.

The invention takes the Mycoplasma hyopneumoniae M129 sequence 16S rDNA gene and the porcine chlamydia ompA gene as detection targets, designs the nested PCR primer group which can simultaneously detect the Mycoplasma hyopneumoniae and the porcine chlamydia in the same PCR reaction program and reaction system, and the Mycoplasma hyopneumoniae detection primer and the porcine chlamydia detection primer cannot be influenced mutually, thereby having the characteristics of strong specificity, high sensitivity and high accuracy and being capable of detecting the pathogen with the copy number as low as a single copy number.

The invention also provides application of the primer group in preparation of products for detecting mycoplasma hyopneumoniae and chlamydia suis.

In the present invention, the product preferably comprises a kit, a reagent and/or a test strip. When the primer group is used for detecting mycoplasma hyopneumoniae and chlamydia suis, the detection method preferably comprises the following steps: extracting DNA of a sample to be detected, carrying out nested PCR, carrying out first round PCR amplification by adopting outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, carrying out second round PCR amplification by adopting inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R, and carrying out electrophoresis detection on a PCR product of each time to detect mycoplasma hyopneumoniae and chlamydia hyopneumoniae.

The invention has no special limitation on the extraction mode of the DNA of the sample to be detected, and can adopt the conventional DNA extraction method in the field. In the present invention, the sample to be tested is preferably a pig blood sample or a tissue sample, or a bronchial lavage, a pig anus swab, or a nose swab sample. The source of the sample to be detected is not particularly limited, for example, a porcine blood sample can be derived from a fetus, a tissue sample can be derived from a porcine lung sample, a porcine lung bronchus sample, a placenta, a lymph node and an mesentery, and the sample to be detected can be semen, a semen supernatant, aerosol and an environmental sample. The invention has no special limitation on the processing mode of each sample to be detected, and can adopt the conventional method for processing the sample to be detected in the field, in the specific embodiment of the invention, when the serum sample is processed, the blood is collected from the anterior vena cava, centrifuged at 1500r/min for 30min, the sample serum is collected, 200 mu L of the sample serum is extracted by using the DNA extraction kit, and the concentration is measured for later use; when the tissue sample is processed, the tissue organ of the sample of the pig to be detected is taken, DNA is extracted by using a DNA extraction kit after the tissue organ is crushed, and the concentration is measured for later use; when the lavage liquid is treated, normal saline is used for lavage of pig lungs, the lavage liquid is collected and centrifuged at 12000r/min for 5min, 200ul of supernatant is collected, DNA is extracted by using a DNA extraction kit, and the concentration is measured for later use; when the anal swab, the nasal swab and the environmental sample are processed, a vortex oscillator is used for oscillating for 2min, the mixture is instantaneously centrifuged for 30s, 200ul of supernatant is taken, DNA is extracted by using a DNA extraction kit, and the concentration is measured for later use.

In nested PCR of the present invention, the outside primer sequences are:

Mhp-O-F: the upstream primer 5'-ACTGACGCTTAGGCTTGAA-3' is the primer that is used,

Mhp-O-R: the downstream primer 5'-TCCCTTCCTTCCTCCAA-3' is a primer that is complementary to the primer,

suis-O-F: the upstream primer 5'-GCCTTATGATTGACGGGATT-3' is the primer that is used,

suis-O-R: a downstream primer 5'-GGTTTAGACTGAGCGTATTGG-3';

the sequence of the inner primer is as follows:

Mhp-I-F: the upstream primer 5'-TTCGCAAGAATGAAACTCA-3' is the primer that is used,

Mhp-I-R: the downstream primer 5'-TCCCTTCCTTCCTCCAA-3' is a primer that is complementary to the primer,

suis-I-F: the upstream primer 5'-CGTCTCGGCTATTATGGA-3' is the primer that is used,

suis-I-R: the downstream primer 5'-CAAGCGAAGGCAGTATCT-3'.

In the present invention, the reaction system of the first round of PCR is preferably 12.5 parts by volume of Taq enzyme, 1 part by volume of each of the outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, 1 part by volume of DNA template, and 1 part by volume of ddH₂And O2.5 parts. The sources of the above substances are not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used.

In the present invention, the reaction conditions of the first round of PCR preferably include: 94 ℃ for 5min, 94 ℃ for 30s, 52-56 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

In the present invention, the reaction system of the second round of PCR is preferably 12.5 parts by volume of Taq enzyme, 1 part by volume of each of the inner primers Mhp-I-F, Mhp-I-R, C.suis-I-F and C.suis-I-R, 1 part by volume of DNA template, and 1 part by volume of ddH₂And O2.5 parts. The sources of the above substances are not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used.

In the present invention, the reaction conditions of the second round of PCR preferably include: 94 ℃ for 5min, 94 ℃ for 30s, 54-58 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min.

In the invention, the existence of mycoplasma hyopneumoniae and chlamydia hyopneumoniae is judged according to the electrophoresis detection result, and the judgment standard is as follows: if a band appears at 418bp or 275bp in the two-round PCR, the mycoplasma hyopneumoniae is positive; if a band appears at 562bp or 395bp, the swine chlamydia is positive; after two rounds of nested PCR, no target band was detected and reported as negative. And after the first round of PCR amplification, the target band is detected through electrophoresis, the report is positive, the toxic amount of the detected sample is high, no target band exists after the first round of PCR amplification, but the target band detected through electrophoresis after the second round of PCR amplification is also judged to be positive, and the toxic amount of the sample is relatively low. The specific method used in the electrophoresis detection of the present invention is not particularly limited, and in the specific embodiment of the present invention, the PCR product is detected by electrophoresis using 1.5% agarose gel.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

2 parallel experiments were performed with the Mycoplasma hyopneumoniae 16S rDNA gene and the porcine chlamydia ompA gene as templates. The steps of the 2 replicates were: carrying out first round PCR amplification by using outer primers Mhp-O-F, Mhp-O-R, C.suis-O-F and C.suis-O-R, wherein the reaction system of the first round PCR is 12.5 mu L of Taq enzyme, and the outer primers

Mhp-O-F：5’-ACTGACGCTTAGGCTTGAA-3’，

Mhp-O-R：5’-TCCCTTCCTTCCTCCAA-3’，

C.suis-O-F：5’-GCCTTATGATTGACGGGATT-3’，

suis-O-R: 5'-GGTTTAGACTGAGCGTATTGG-3' mu.L each, DNA template 1. mu.L, ddH₂O2.5. mu.L, the reaction conditions of the first round of PCR are: 94 ℃ for 5min, 94 ℃ for 30s, 52-56 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min. The PCR product was detected by electrophoresis on a 1.5% agarose gel, and a second amplification was performed if the target band was not present.

Using the first round amplification product as a template and an inner primer

Mhp-I-F：5’-TTCGCAAGAATGAAACTCA-3’，

Mhp-I-R：5’-TCCCTTCCTTCCTCCAA-3’，

C.suis-I-F：5’-CGTCTCGGCTATTATGGA-3’，

suis-I-R: 5'-CAAGCGAAGGCAGTATCT-3' second round of amplification, the second round of PCR reaction system is Taq enzyme 12.5. mu.L, inner primers Mhp-I-F, Mhp-I-R and C.suis-I-F, C.suis-I-R are each 1. mu.L, DNA template 1. mu.L, ddH₂O2.5. mu.L, and the reaction conditions of the second round of PCR are as follows: 94 ℃ for 5min, 94 ℃ for 30s, 54-58 ℃ for 30s, 72 ℃ for 45s, 32 cycles, 72 ℃ for 5 min. The PCR product was detected by electrophoresis on a 1.5% agarose gel. The results of the detection are shown in FIG. 1.

As can be seen from FIG. 1, the target bands detected by the outer primers are 562bp and 418bp, and the target bands detected by the inner primers are 275bp and 395 bp.

Example 2

The primers and primers are characterized in that genes for extracting positive swine (positive control) of mycoplasma hyopneumoniae and swine chlamydia, hog cholera virus, porcine parvovirus and porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, escherichia coli and staphylococcus aureus are respectively used as templates, nested PCR is carried out, sterile distilled water is used as negative control, the specific steps of nested PCR are the same as example 1, and electrophoresis detection is carried out on PCR products by using 1.5% agarose gel. The results are shown in FIG. 2.

As can be seen from FIG. 2, the four primer sequences used in the nested PCR designed by the present invention have higher specificity.

Example 3

Extracting Mhp and C.suis plasmid, measuring concentration and calculating copy number to 5.0 × 10⁹Then, 10-fold dilution is carried out to the single-digit copy number, each dilution plasmid is selected as a template, nested PCR is carried out, the specific steps of nested PCR are the same as example 1, and the PCR product is detected by electrophoresis with 1.5% agarose gel. The results are shown in FIG. 3, and the lowest 5.0X 10 detected after two rounds of nested PCR amplification⁰Number of copies.

Example 4

The swine mycoplasma and chlamydia recombinant plasmids are respectively mixed with the classical swine fever virus, the porcine parvovirus, the porcine pseudorabies virus and the escherichia coli in equal amount, the sterile non-enzyme water is used as negative control, nested PCR is carried out, the specific steps of the nested PCR are the same as the example 1, the PCR product is subjected to electrophoresis detection by 1.5 percent agarose gel, the anti-interference performance of the primer is checked, and whether a target band can be amplified from the mixed genome template is observed. The results are shown in FIG. 4, the specific bands can be amplified from the mixed template by both the inner primer and the outer primer, and the primers are shown to have better anti-interference performance.

Example 5

20 parts of piglet blood samples are collected from a scaled pig farm in Hubei in 2021 month, centrifuged at 1500r/min for 30min, sample serum is collected, DNA is extracted according to a column type blood sample DNA extraction kit of Kangji century company as a template, nested PCR is carried out, the specific steps of the nested PCR are the same as those in example 1, and the PCR products are subjected to electrophoresis detection by using 1.5% agarose gel. The result shows that 15 positive samples are detected in 20 serum samples, wherein 10 mycoplasma are detected, 5 chlamydiae are detected, and the sequencing result shows that 15 positive products are mycoplasma hyopneumoniae genes and porcine chlamydia genes. The method of the invention is proved to have reliability in detection result.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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