CN113718044A

CN113718044A - Primer, probe and kit for fluorescent quantitative detection of swine mycoplasma, mycoplasma microformis and new mycoplasma haemophilus

Info

Publication number: CN113718044A
Application number: CN202010452628.0A
Authority: CN
Inventors: 付媛; 石团员; 徐丽华; 袁秀芳; 孙洪超; 李军星; 韦强; 王荣申
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2021-11-30

Abstract

The invention discloses a primer, a probe and a kit for fluorescent quantitative detection of mycoplasma hyopneumoniae, mycoplasma microminiae and new mycoplasma haemophilus. The primer and the probe are shown in a sequence table SEQ ID NO: 1 to SEQ ID NO: shown at 9. The invention also discloses a fluorescent quantitative PCR detection kit containing the primer and the probe. The detection kit can be used for simultaneously detecting and distinguishing 3 important porcine haemophilus infections, realizes one-time sampling and one-time analysis, and provides technical support for the research, prevention and control of the disease.

Description

Primer, probe and kit for fluorescent quantitative detection of swine mycoplasma, mycoplasma microformis and new mycoplasma haemophilus

Technical Field

The invention belongs to the technical field of molecular detection, and particularly relates to a primer, a probe and a kit for fluorescent quantitative detection of mycoplasma hyopneumoniae, mycoplasma microminia and mycoplasma haemophilus.

Background

Porcine haemophilus (formerly known as Eperythrozoonosis) is a disease that mycoplasma Haemophilus (HM) parasitizes on the surface of pig red blood cells and in bone marrow, causes infectious anemia, subsequently causes stillbirth and abortion of sows, and can also cause immune suppression of organisms, and causes death of pigs by being combined with other pathogens and secondary infection. The swine haemophilus mycoplasma disease is gradually in a wide epidemic trend since the swine haemophilus mycoplasma disease is first discovered in Jiangsu of China in the eighties of the last century, and the epidemic range of the swine haemophilus mycoplasma disease almost extends to the whole nation at present.

Three types of HM have been reported at home and abroad for infected pigs, Mycoplasma hyopneumoniae (M.suis), Mycoplasma parvum (M.parvum), and the isolation of a new species of Mycoplasma haemophilus (CMh) in Zhejiang. Clinical investigation shows that many cases of mixed infection occur, but the three pathogens cannot be identified by the existing PCR detection method and standard.

The invention utilizes the characteristics of specificity, sensitivity and the like of the fluorescent PCR technology in the aspect of nucleic acid detection, establishes a gapA gene-based fluorescent quantitative PCR detection method, can simultaneously quantitatively identify three swine haemophilus infections, and provides technical support for the research and prevention and control of the disease.

Disclosure of Invention

The first purpose of the invention is to provide a group of primers and probes for fluorescence quantitative PCR, which have strong specificity and high sensitivity and are used for simultaneously detecting mycoplasma hyopneumoniae (M. suis), mycoplasma parvum (M.parvum) and mycoplasma haemophilus (CMh).

Another objective of the present invention is to provide a rapid, accurate and easy-to-use fluorescence quantitative PCR detection kit and method for simultaneously detecting mycoplasma hyopneumoniae (m.suis), mycoplasma microminia (m.parvum) and mycoplasma neohaemophilus (CMh).

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

according to the nucleotide sequence registered by GenBank, the gene sequences of M.suis (registration number KU246053), M.parvum (registration number KU246052) and CMh (registration number KU246051) glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase GAPDH) are selected, and the variation region is selected as the amplification target sequence region through the comparison analysis of bioinformatics software. Primer Premier 5.0 and Beacon Designer 4 are used for respectively designing a plurality of pairs of primers and specific probes, experimental screening is carried out, and finally 3 pairs of primers and 3 probes are determined, wherein the sequences are shown in Table 1, wherein the M.suis probe is marked by 2, 7-dimethyl-4, 5-dichloro-6-carboxyfluorescein (JOE), the M.parvum probe is marked by CY5(Cy5), the CMh probe is marked by 5-carboxyfluorescein (FAM), and the primers and the probes are synthesized by Shanghai Biotech limited.

TABLE 1 fluorescent quantitative PCR primers and probes for M.suis, M.parvum and CMh

A or G is the code of degenerate basic group; y is C or T; m is A or C; k is G or T; w is A or T

The invention also provides a fluorescent quantitative PCR detection kit for simultaneously detecting the mycoplasma hyopneumoniae (M.suis), the mycoplasma parvum (M.parvum) and the new mycoplasma haemophilus (CMh).

The kit comprises the primer and the probe for the fluorescent PCR detection. Further, the kit may further comprise materials and reagents required to complete the fluorescent quantitative PCR reaction, for example, reaction buffer, enzymes, positive controls, negative controls, and the like.

In one embodiment of the invention, the positive controls are plasmids containing the genes for Mycoplasma hyopneumoniae gapA, Mycoplasma microminiae gapA and Mycoplasma new-type gapA, respectively.

The invention also provides a fluorescent quantitative PCR method for simultaneously detecting the mycoplasma hyopneumoniae, the mycoplasma microminiae and the new mycoplasma haemophilus, which comprises the following steps:

(1) extracting DNA from blood;

(2) preparing a PCR reaction system, wherein the reaction system comprises the primer pair and the probe;

(3) carrying out fluorescence PCR reaction;

(4) and (6) judging the result.

Specifically, the concentration corresponding to the Ct value of the mycoplasma hyopneumoniae (M.suis) and the new mycoplasma haemophilus (CMh) is less than 35, the Ct value of the mycoplasma mimutum (M.parvum) is less than 34, and the Ct value of the S-shaped amplification curve is taken as the detection result.

The invention has the advantages that: can quantify, detect pathogen infection intensity, can detect three pig source hemophilus mycoplasma pathogens simultaneously, sensitivity is 100 times higher than common PCR, reaches 10 copies.

Drawings

FIG. 1: and (3) performing fluorescence quantitative PCR amplification electrophoresis result on the target fragment, wherein M: pUC19 DNA/MspI (HpaII) Marker; 1. negative control, 2.CMh single TaqMan qPCR fragment of interest; m.parvum single TaqMan qPCR target fragment; m.suis single TaqMan qPCR target fragment; 5. triple TaqMan qPCR fragments of interest.

FIG. 2: triple TaqMan probe real-time fluorescence quantitative PCR standard curve.

FIG. 3: triple TaqMan probe qPCR sensitivity test amplification curve.

FIG. 4: a triple TaqMan probe qPCR specificity test amplification curve; wherein, 1: a swine mycoplasma; 2: a mycoplasma microforme; 3: a new species of mycoplasma haemophilus; 4: erysipelas of pig; 5: e.coli; 6: salmonella; 7: mycoplasma hyorhinis; 8: mycoplasma hyopneumoniae; 9: haemophilus parasuis; 10: mycoplasma bovis; 11: and (5) negative control.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The methods used in the following examples are conventional methods unless otherwise specified, and the test materials used are commercially available from conventional biochemical reagent suppliers unless otherwise specified.

Strains and clinical samples

Coli TOP 10 competent cells were purchased from Beijing Quanjin Biotechnology Limited, and porcine-derived M.suis, M.parvum, CMh and M.bovis (Mycoplasma wenyonii M.wenyonii) DNA samples were isolated and stored in the laboratory; haemophilus parasuis (haemophilus parasuis h. parasuis), Escherichia coli (Escherichia coli e. coli), swine erysipelas (erysipelas rhusiopathiae e. rhusiopathiae), Salmonella of swine origin (Salmonella, Mycoplasma hyorhinis (Mycoplasma hyorhinis m. hyorhorinis), Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae m. hyopneumoniae) DNA were kept away from the porcine pathology group at the institute of veterinary medicine of farming in the department of zhejiang; mycoplasma ovis (m.ovis) DNA was gifted by the wangxing doctor of south-river agricultural university. 150 parts of anticoagulated blood sample of binary hybrid sow without obvious symptoms are collected in 2 pig farms in Zhejiang province in 2019.

Reagents and carriers

200 μ L of 8-tubes were purchased from Hangzhou Chuangzhi Biotech Co., Ltd, ExTaq^TMHot Start, dNTP, pMD19-T vector and the like are all purchased from Takara Shuzo (Dalian) Co., Ltd., and pUC19 DNA/MspI (HpaII) Marker and gel recovery and purification kit are purchased from Shanghai Biotechnology Co., Ltd. The whole blood DNA extraction kit is purchased from Hangzhou Xinjing biotechnology development Co.

Example 1 design and Synthesis of primers and probes

Example 2 kit composition

1. Composition of the kit (stored at-20 ℃ C.)

(1) Primers and probes designed in example 1 for detection of mycoplasma hyopneumoniae (m.suis), mycoplasma microminia (m.parvum) and mycoplasma neohaemophilus (CMh);

(2) buffer (MgCl free)₂): purchased from bando (Dalian) limited, cat # 9512 AM;

(3)Ex Taq^TMhot Start: purchased from Baoyi (Dalian) Ltd., cat # RR 006B;

(4)MgCl₂: purchased from bando (Dalian) limited, cat # 9512 AM;

(5) dNTP: product number 4030 from Baoyi (Dalian) Co., Ltd;

(6) ROX reference Dye II: purchased from Baoyi (Dalian) Ltd, cat # RR 390A;

(7) negative control: RNase Free ddH₂O；

(8) Positive control: standard plasmids respectively containing mycoplasma hyopneumoniae gapA gene, mycoplasma microminiae gapA gene and new mycoplasma haemophilus gapA gene;

(9)RNase Free ddH₂o: from Baoybio (Dalian) Co., Ltd., Cat. No. 9012.

2. Standard plasmid preparation

The methods for extracting m.suis, m.parvum and CMh DNA in this experiment were performed with reference to the instructions of the blood DNA extraction kit (new scenery, hangzhou). Three HM gapA full-length genes are amplified by PCR, and amplification primers are shown in a table 1 and are shown in a sequence table SEQ ID NO: 10 to SEQ ID NO: shown at 15. The reaction system is as follows: the 25. mu.L system included 2.5. mu.L of 10 XBuffer (containing MgCl)₂) mu.M dNTPs, 1U of ExTaq DNA polymerase, 250. mu.M of the primers msgf/msgr, mpgf/mpgr or cmhgf/cmhgr (primer sequences are shown in Table 1), 5. mu.L of template DNA, and the remainder filled with water. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 94 ℃ for 30sec, annealing at 45 ℃ for 1min, extension at 72 ℃ for 1min, 35 cycles in total, and extension at 72 ℃ for 10 min. The PCR product is purified and recovered by a glue recovery and purification kit (raw, Shanghai).

TA cloning, wherein a purified product is connected with a pMD18-T vector (Baozhi, Dalian) according to a DNA Ligation Kit, transformed into Escherichia coli (Escherichia coli) TOP 10 competent cells, cultured for 18h at 37 ℃, and subjected to colony PCR screening, positive cloning and sequencing identification by a company (Sheng, Shanghai). Positive plasmids were identified, concentration determined, stored at-20 ℃ as standard for future use, according to the formula: copy/. mu.L ═ (ng/. mu.L. times.10)^-9)×(6.02×10²³) /(DNA length. times.660) the plasmid concentration was converted to copy number, respectively.

Example 3 establishment of detection method

(1) Triple TaqMan probe fluorescent quantitative PCR method and reaction system optimization

First, the enzyme and Mg were optimized separately²⁺Ion concentration: ExTaq was optimized in a 20. mu.L reaction system^TMAdjusting the Hot Start enzyme in a gradient of 1.25-5U, MgCl₂The concentration is adjusted in a gradient of 2.5-5 mM, Buffer (MgCl is not contained)₂) mu.L, 0.4. mu.L each of the upstream and downstream primers (10. mu. mol/L) M.suis, M.parvum and CMh and 0.8. mu.L each of the probes msgp577, mpgp129 and cmhgp217 (10. mu. mol/L), 2.5mM dNTP 2. mu.L, 0.2. mu.L of ROX reference Dye II (50X), 2. mu.L of template, the remainder being filled with water. The amplification conditions were: 30s at 95 ℃; then, 40 cycles of 95 ℃ for 5s and 60 ℃ for 34s are carried out, and the enzyme and Mg with the earliest Ct value and high relative fluorescence value of an amplification curve are screened²⁺The ion concentration.

Secondly, optimizing the concentration of the primer and the probe by adopting a matrix method, wherein in a 20 mu L reaction system: buffer (MgCl not contained)₂)2 mu L, screening to the optimal exTaq^TMHot Start enzyme concentration and MgCl₂The concentration of primers and probes (10. mu. mol/L) at upstream and downstream of M.suis, M.parvum and CMh was adjusted to 0.4 to 1.0. mu.L, 2. mu.L of 2.5mM dNTP, 0.2. mu.L of ROX reference Dye II (50X), 2. mu.L of template, and the balance was made up with water. The amplification conditions were: 930s at 5 ℃; and then, carrying out 40 cycles at 95 ℃ for 5s and 60 ℃ for 34s, and screening out the optimal matching concentration of the primers and the probes, which has less influence on amplification efficiency and lower use cost, and can simultaneously detect M.suis, M.parvum and CMh before 35 cycles according to the result.

As a result: according to the single TaqMan fluorescent quantitative PCR for establishing M.suis, M.parvum and CMh and the optimization of the primer and probe concentration and the reaction system by the matrix method, the optimal reaction system of 20 uL is finally determined as shown in Table 2. The amplification conditions were: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 60 ℃ for 34s, 40 cycles, and reaction on ABI7500 real-time fluorescence quantitative PCR instrument. The electrophoresis results of the amplified products are shown in FIG. 1.

TABLE 2 triple TaqMan fluorescent quantitative PCR reaction systems M.suis, M.parvum and CMh

Reagent	Addition amount/. mu.L
		Buffer (Mg)²⁺ free)(10X)	2
Ex Taq(HS)(5U/μL)	0.5
		MgCl₂(25mM)	2.8
dNTP(2.5mM)	2
		msgf556and rmsgr717(10μM)	0.6
mpgf16and mpgr155(10μM)	0.8
		cmhf157and cmhr256(10μM)	0.6
TaqMan^@msProbe JOE(10μM)	0.6
		TaqMan^@mpProbe CY5(10μM)	0.4
TaqMan^@cmhProbe FAM(10μM)	0.8
		ROX reference Dye II(50X)	0.2
DNA template	2
		Water RNase Free ddH₂O	6.7

(2) Establishment of standard substance curve of triple TaqMan probe fluorescent quantitative PCR method

Plasmid standards of M.suis, M.parvum and CMh were mixed at known copy numbers and serially diluted 10-fold with Easy Dilution to form a concentration gradient (1.0X 10)⁸～1.0×10^-1copies/. mu.L) under the optimized conditions and amplification system obtained at 1.5, and the number of copies of the positive standard as the abscissa and the number of cycles (Ct value) detected at each concentration as the ordinateThree swine mycoplasma hyorhinis TaqMan probe PCR standard curves are established according to coordinates.

Plasmid standards M.suis, M.parvum and CMh were expressed at 1.0X 10⁸The copies/mu L is used as the original time to carry out 10 times of gradient dilution, and 1.0 multiplied by 10 is taken⁸The 9 concentration-gradient standard sample of 1.0X 100 copies/. mu.L was amplified according to the reaction system and reaction conditions optimized at 1.6, and a standard curve was drawn. See fig. 2 for the correlation coefficient R of the standard curve of m.suis²0.9993, and the amplification efficiency E is 103.2%; correlation coefficient R of standard curve of M.parvum detection²0.9992, the amplification efficiency E is 99.9%; and CMh, the correlation coefficient R of the standard curve²When the amplification efficiency was 0.9996, the amplification efficiency E was 103.0%.

(3) Triple TaqMan probe fluorescent quantitation PCR sensitivity assay

A mixed plasmid standard of M.suis, M.parvum and CMh of known copy number was diluted to a concentration of 1.0X 10⁸～1.0×10^-2And (3) performing fluorescent quantitative PCR amplification by using copies/mu L as a template and using the optimized condition and the amplification system obtained in the step (1), repeating 3 samples in each concentration gradient, determining the cycle number (Ct value) at each concentration, and using the lowest dilution concentration at which the Ct value is less than 35 cycles and an S-shaped amplification curve appears as the detection sensitivity.

A mixed plasmid standard of M.suis, M.parvum and CMh of known copy number was diluted to a concentration of 1.0X 10⁸～1.0×10^-2The copies/. mu.L is used as a template for amplification, and as shown in the result of FIG. 3, the detection lower limit of the triple TaqMan probe fluorescent quantitative PCR method established by the invention for M.suis, M.parvum and CMh is 10 copy/. mu.L.

(4) Triple TaqMan probe fluorescent quantitative PCR specificity test

Using 1.5 optimized system and conditions to perform fluorescent quantitative PCR amplification on three porcine HM, DNA of other mycoplasma haemophilus and bacteria such as M.wenyonii, M.ovis, H.parasuis, E.coli, E.rhesiopathiae, Salmonella, M.hyorhinis and M.hyopneumoniae as templates, setting positive and negative controls, and using the optimized conditions and amplification system obtained in the step (1) to check the specificity of the research method.

The optimized system and conditions in the step (1) are used for detecting other mycoplasma and porcine bacteria such as M.wenyonnii, M.ovis, H.parasuis, E.coli, E.rhosiopathiae, Salmonella, M.hyorhinii and M.hyopneumniae, and negative and positive controls are set, so that the result shows that except the specific amplification curves generated by M.suis, M.parvum and CMh, the amplification curves generated by other pathogens and deionized water are not generated (figure 4).

(5) Triple TaqMan probe fluorescent quantitative PCR repeatability test

Negative controls were set at 3 known concentrations (1.0X 10)⁴、1.0×10⁵、1.0×10⁶copies/. mu.L) of the three HM standard plasmid mixed samples as templates, each sample was repeated 6 times, and the Coefficient of Variation (CV) in the cyclic array was calculated at 3 known concentrations (1.0X 10)⁴、1.0×10⁵、1.0×10⁶copies/mu L) as a template, repeating 1 time every 3 days, repeating 3 times per sample for detection, and calculating the Coefficient of Variation (CV) of the cycle number to verify the repeatability among groups.

At 3 known concentrations (1.0X 10)⁴、1.0×10⁵、1.0×10⁶copies/. mu.L) of three HM standard plasmid mixed samples as templates, and performing repeatability tests under (1) optimized conditions. Repeating for 1 time at intervals of 3 days, and repeating for 3 times to perform repeated measurement among groups; each group was replicated 6 times for an intra-group replicate assay. The results show that the Coefficient of Variation (CV) of the number of cycles of the sample detection between the groups is between 0.31 and 1.75 percent (Table 3), and the detection method has better repeatability.

TABLE 3 triple TaqMan fluorescent quantitation PCR reaction repeatability test

Example 4: detection of clinical samples by kit

The triple TaqMan probe fluorescent quantitative PCR method is adopted to detect 118 anticoagulant samples in 2019-2020 years clinically and simultaneously detect the samples by the established double PCR method, the results are shown in Table 4, the conventional double PCR method detects that the M.suis/M.parvum positive rate is 34.7 percent (41/118), the detected CMh positive rate is 5.9 percent (7/118), and the detected M.suis/M.parvum and CMh pathogen mixed infection positive rate is 2.5 percent (3/118); the triple TaqMan probe fluorescence quantitative PCR detection method detects that the M.suis positive rate is 44.1% (52/118), the M.parvum positive rate is 41.5% (49/118), the positive rate of the detected CMh is 43.2% (51/118), the positive rate of the detected M.suis and M.parvum pathogen mixed infection is 14.4% (17/118), the positive rate of the detected three pathogen mixed infection is 19.5% (23/118), and the detection results of the triple TaqMan probe qRPCR of a sample detected to be positive by the double conventional PCR are positive, which indicates that the triple TaqMan probe qPCR detection method is more sensitive than the conventional PCR method.

TABLE 42 comparison of results of detection of porcine haemophilus

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> primer, probe and kit for fluorescent quantitative detection of swine mycoplasma, mycoplasma microformis and new mycoplasma haemophilus

<130> JLP20I0244

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> artificially synthesized sequence msgf556

<400> 1

catgctttca cttctgac 18

<210> 2

<211> 18

<212> DNA

<213> artificially synthesized sequence msgr717

<400> 2

gtgtgcaatt ccatcaag 18

<210> 3

<211> 23

<212> DNA

<213> artificially synthesized sequence msgp577

<400> 3

agacttcaag actctcctca cgc 23

<210> 4

<211> 20

<212> DNA

<213> artificially synthesized sequence mpgf16

<400> 4

gtagcaatta atggtttgg 19

<210> 5

<211> 19

<212> DNA

<213> artificially synthesized sequence mpgr154

<400> 5

gcgcactatc atactttag 19

<210> 6

<211> 25

<212> DNA

<213> artificially synthesized sequence mpgp129

<400> 6

cgctaacact ccaatatcaa ccaca 25

<210> 7

<211> 19

<212> DNA

<213> artificially synthesized sequence cmhgf157

<400> 7

tgaggttgag atgtttctg 19

<210> 8

<211> 20

<212> DNA

<213> artificially synthesized sequence cmhgr256

<400> 8

ccctttcaga acaaacctta 20

<210> 9

<211> 27

<212> DNA

<213> artificially synthesized sequence cmhgp217

<400> 9

tacaactaca tccacacttc acaacca 27

<210> 10

<211> 23

<212> DNA

<213> artificially synthesized sequence msgf

<400> 10

atgacaatcc acaaagtagc aat 23

<210> 11

<211> 28

<212> DNA

<213> artificially synthesized sequence msgr

<400> 11

ttaaagagaa atgtagtatt tcaaagtt 28

<210> 12

<211> 22

<212> DNA

<213> artificially synthesized sequence mpgf

<400> 12

atgrmaatyy aaragtagca at 22

<210> 13

<211> 23

<212> DNA

<213> artificially synthesized sequence mpgr

<400> 13

ttaaagagaa ryrwagtart kra 23

<210> 14

<211> 25

<212> DNA

<213> artificially synthesized sequence cmhgf

<400> 14

atgaaaaata tagctattaa cggtt 25

<210> 15

<211> 27

<212> DNA

<213> artificially synthesized sequence cmhgr

<400> 15

ttaaagagaa gcgaaatgac aaacagt 27

Claims

1. A set of reagents for simultaneously detecting mycoplasma hyopneumoniae, mycoplasma microminiae and mycoplasma neohaemophilus, wherein the reagents comprise the following primer pairs and probes:

the primer pair for detecting the mycoplasma hyorhinis is shown as a sequence table SEQ ID NO: 1 to SEQ ID NO: 2, and the probe for detecting the swine mycoplasma is shown in a sequence table SEQ ID NO: 3 is shown in the specification;

the primer pair for detecting the mycoplasma parvum is shown as the sequence table SEQ ID NO: 4 to SEQ ID NO: 5, and the probe for detecting the mycoplasma parvum is shown as a sequence table SEQ ID NO: 6 is shown in the specification;

the primer pair for detecting the new mycoplasma haemophilus is shown as the sequence table SEQ ID NO: 7 to SEQ ID NO: 8, and the probe for detecting the new mycoplasma haemophilus is shown as a sequence table SEQ ID NO: shown at 9.

2. The kit according to claim 1, wherein the probe for detecting swine mycoplasma is labeled with JOE at the 5 'end and BHQ1 at the 3' end; the probe for detecting the mycoplasma microtuberis is marked with CY5 at the 5 'end and BHQ2 at the 3' end; the 5 'end of the probe for detecting the new mycoplasma haemophilus is marked with FAM, and the 3' end of the probe is marked with BHQ 1.

3. A kit for simultaneously detecting mycoplasma hyopneumoniae, mycoplasma microminiae and mycoplasma neohaemophilus, comprising a kit of parts according to claim 1 or 2.

4. The kit of claim 3, further comprising PCR reaction buffer, enzyme, positive control, and negative control.

5. The kit of claim 4, wherein the positive control is a plasmid containing the genes Mycoplasma hyopneumoniae gapA, Mycoplasma microminiae gapA and Mycoplasma neohaemophilus gapA, respectively.

6. A method for simultaneously detecting mycoplasma hyopneumoniae, mycoplasma microminia and a new mycoplasma haemophilus is characterized in that: the method comprises the following steps:

(1) extracting DNA from blood;

(2) preparing a PCR reaction system, wherein the reaction system comprises the kit of the reagent set of claim 1 or 2;

(3) carrying out fluorescence PCR reaction;

(4) and (6) judging the result.