CN107227380A - The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection - Google Patents

Abstract

The invention discloses two pairs of specific primers and a composite PCR detection method for synchronous detection of PCV2 and PRV, wherein the two pairs of specific primers have equivalent GC content, similar annealing temperature, strong specificity, no primer dimer, and The amplified PCV2 and PRV fragments are of appropriate size and can be clearly distinguished in agarose gel electrophoresis, which is convenient for intuitive observation of experimental results. The present invention detects PCV2 and PRV synchronously through reasonable setting of various technical parameters of the composite PCR, which is simple, convenient, formulaizable, low in technical requirements, low in cost, time-saving and labor-saving, and ensures good specificity, sensitivity and stability , suitable for clinical application.

Description

A kind of primer sequence and method for simultaneous detection of PCV2 and PRV infection

technical field

The invention relates to the field of biotechnology, in particular to a primer sequence and a method for synchronous detection of PCV2 and PRV mixed infection.

Background technique

Porcine circovirus type 2 (PCV2) and porcine pseudorabies virus (PRV) infection are widespread and serious DNA virus diseases in pig production in my country. Among them, the mortality rate of PCV2 can reach more than 50% when it breaks out, so it has become the most important infectious disease in my country's pig industry. PRV belongs to the αherpesvirus subfamily of the family Herpesviridae, and its infection can cause abortion, stillbirth, mummification, acute death of newborn piglets, and neurological symptoms and high mortality in piglets under three weeks of age in pregnant sows; The seropositive rate of pseudorabies in the field reaches more than 70%, and the harm is serious. The above two pathogens are relatively common in clinical practice, and are often co-infected, and the diseases caused by them have caused serious economic losses to the pig industry.

Traditional PCV2 infection and PRV infection detection methods include virus isolation, experimental animal inoculation, and serological tests. These methods are cumbersome to operate, have long detection cycles, and some have disadvantages such as non-specific reactions and low sensitivity, which can no longer meet the needs of breeding. industry's disease prevention needs. With the development of molecular biology, PCR detection method can quickly and efficiently detect pathogens.

Polymerase chain reaction (PCR) is a method of enzymatically synthesizing specific DNA fragments in vitro. It consists of several steps such as high-temperature denaturation, low-temperature annealing (renaturation), and suitable temperature extension. Amplification has the characteristics of strong specificity, high sensitivity, easy operation, and short detection cycle. It can be used not only for basic research such as gene isolation, cloning, and nucleic acid sequence analysis, but also for disease diagnosis.

Conventional PCR is simple to operate and has low technical requirements, but multiple PCR tests are required for multiple virus mixed infections, which in turn prolongs the detection cycle and increases the cost of detection. Multiplex PCR refers to the method of adding multiple pairs of primers to the same PCR reaction system to amplify multiple target genes at the same time; using this method to simultaneously detect multiple target genes saves time, effort and cost, but the primers in the reaction system are very large. It is easy to form complex primer-dimers, which will affect the sensitivity and accuracy of detection. Therefore, the key to the success of multiplex PCR lies in the specificity of designed primers, the optimization of reaction system and procedures.

In the prior art, there is a method for detecting various viruses of pigs by multiple RT-PCR, but it has high requirements for RNA concentration and quality, and is very susceptible to contamination by genomic DNA and RNase during the detection process. Operators have high technical requirements, which is not conducive to widespread application. In addition, multiplex fluorescent PCR detection methods also exist, but the probes labeled with fluorophores with different emission wavelengths are likely to interfere with each other, thereby affecting specific detection.

It can be seen that there is still a lot of room for development in the method for synchronous detection of PCV2 and PRV infection, and those skilled in the art urgently need to design a PCV2 method that is easy to operate, low in technical requirements, high in specificity, strong in stability, and good in repeatability. , PRV synchronous detection method.

Contents of the invention

Based on the shortcomings of the prior art, the present invention provides a method capable of simultaneously detecting mixed infection of PCV2 and PRV.

The technical scheme that the present invention takes in order to realize the above object is:

A primer sequence for simultaneous detection of PCV2 and PRV infection, characterized in that: it includes a PCV2 viral primer sequence and a PRV viral primer sequence; wherein the PCV2 viral primer sequence includes an upstream primer PCV2-F1 and a downstream primer PCV2-F2; the PRV virus primer sequence comprises upstream primer PRV-F3 and downstream primer PRV-F4; The nucleotide sequence of described PCV2-F1, PCV2-F2, PRV-F3 and PRV-F4 is as sequence table SEQ ID NO.1, SEQ ID NO.1 respectively Shown in ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

A method for synchronous detection of PCV2 and PRV infection, characterized in that it comprises the following steps:

(1) design two pairs of specific primers as shown in claim 1 according to the nucleotide sequence design of PCV2ORF2 gene and PRV gH gene registered in GenBank;

(2) Use the two pairs of specific primers to carry out compound PCR amplification on the sample to be tested to obtain a PCR product, which is detected by agarose gel electrophoresis; if a band of 470bp size appears, the sample to be tested contains PCV2 virus; if If a band with a size of 298bp appears, the sample to be tested contains PRV virus.

Further, the reaction system of the multiplex PCR amplification is 50.0 μL, including: 10×Buffer 5.0 μL, 2.0 mM MgCl ₂ 4.0 μL, 200 μM dNTPs 5.0 μL, 0.1 μM PCV2-F11.0 μL, 0.1 μM PCV2-F21. 0 μL, 0.2 μM PRV-F3 2.0 μL, 0.2 μM PRV-F4 2.0 μL, rTaq DNA polymerase 0.5 μL, DNA template 2.0 μL, ddH ₂ O 27.5 μL.

Further, the reaction program of the multiplex PCR amplification is 95°C for 5 minutes; 94°C for 30 s, 56°C for 30 s, 72°C for 45 s, 35 cycles; finally, 72°C for 10 min.

Beneficial effects: the invention provides two pairs of specific primers and a composite PCR detection method for synchronous detection of PCV2 and PRV. By comparing the gene common characteristics of 15 PCV2 virus strains and 18 PRV virus strains in the GenBank database and the nucleotide sequences of PCV2 ORF1 and PRV gH genes, two pairs of specific primers were designed using Primer Premier software. Relatively, the annealing temperature is similar, the specificity is strong, and primer dimers will not be generated, and the amplified PCV2 and PRV fragments are of appropriate size, which can be clearly distinguished in agarose gel electrophoresis, which is convenient for visually observing the experimental results. The present invention detects PCV2 and PRV synchronously through reasonable setting of various technical parameters of the composite PCR, which is simple, convenient, formulaizable, low in technical requirements, low in cost, time-saving and labor-saving, and ensures good specificity, sensitivity and stability , suitable for clinical application.

Description of drawings

Fig. 1 shows composite PCR amplification test result of the present invention; Among the figure, M is pUC19 DNA/Msp1 Marker, and 1 is PCV2 DNA amplification product; 2 is PRV DNA amplification product; The amplification of PCV2 DNA and PRV DNA mixed template product.

Fig. 2 shows the specificity test result of compound PCR of the present invention; Among the figure, M is pUC19 DNA/Msp1 Marker, and 1 is the amplification product of PCV2 DNA and PRV DNA mixed template; 2 is PPV DNA amplification product; 3 is ETEC DNA amplification product; 4 is APP DNA amplification product.

Fig. 3 shows the sensitivity test result of the composite PCR of the present invention; M in the figure is pUC19 DNA/ ^Msp1 Marker, 1-8 is the amplified product under ^10-1-10-8 dilution factor of PCV2 DNA and PRV DNA mixed template.

detailed description

Below in conjunction with embodiment the present invention is further described in detail:

Example 1

(1) Design and synthesis of primers

By comparing the gene common features of 15 PCV2 virus strains and 18 PRV virus strains in the GenBank database and the nucleotide sequences of PCV2 ORF1 (SEQ ID NO.5) and PRV gH (SEQ ID NO.6) genes, Primer Premier software designed two pairs of specific primers, including PCV2 virus primers and PRV virus primers.

Wherein, PCV2 virus primer comprises upstream primer PCV2-F1 and downstream primer PCV2-F2; PRV virus primer comprises upstream primer PRV-F3 and downstream primer PRV-F4; PCV2-F1, PCV2-F2, PRV-F3 and PRV-F4 The nucleotide sequences are shown in Table 1, respectively. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the sizes of the amplified products are expected to be 470bp and 298bp, respectively.

Table 1

(2) Extraction of viral genomic DNA

Take 0.1 g of PCV2 and PRV-positive disease materials respectively, put them in Eppendorf tubes, add 1 ml of TE buffer solution, twist them into a homogenate with a twister, freeze and thaw three times at -70 ° C, centrifuge to get 0.2 ml of the supernatant, and then use the virus Viral DNA was extracted with the DNA extraction kit, and finally eluted in 50 μl TE buffer and stored at -20°C.

(3) Composite PCR detection;

Use PCV2DNA or PRV DNA or a 1:1 mixture of PCV2DNA and PRV DNA as a DNA template for multiplex PCR amplification. The reaction system for multiplex PCR amplification is 50.0μL, including: 10×Buffer 5.0μL, 2.0mM MgCl ₂ 4.0μL, 200 μM dNTPs 5.0 μL, 0.1 μM PCV2-F11.0 μL, 0.1 μM PCV2-F2 1.0 μL, 0.2 μM PRV-F3 2.0 μL, 0.2 μM PRV-F4 2.0 μL, rTaq DNA polymerase 0.5 μL, DNA template 2.0 μL, ddH ₂ O 27.5 μL.

The reaction program of the multiplex PCR amplification was 95°C for 5min; 94°C for 30s, 56°C for 30s, 72°C for 45s, 35 cycles; finally, 72°C for 10min, and stored at 4°C for later use.

After the reaction, the PCR amplification products were detected by agarose gel electrophoresis, and the detection results are shown in FIG. 1 . A clear target band was amplified in lane 1, about 470bp, which was equivalent to the expected size; after the PCR product was purified and recovered, it was connected to the pGEM-T vector for cloning, transformation, and sequencing. Sequence analysis showed that the cloned target band The size of the DNA fragment is 470bp, which is the target DNA fragment expected to be amplified, which proves that the sample contains PCV2.

A clear target band was amplified in lane 2, about 298bp, which was equivalent to the expected size; after the PCR product was purified and recovered, it was connected to the pGEM-T vector for cloning, transformation, and sequencing. Sequence analysis showed that the cloned target band The size of the DNA fragment is 298bp, which is the target DNA fragment expected to be amplified, which proves that the test sample contains PRV.

Two clear target bands were simultaneously amplified in lane 3, about 470bp and 298bp respectively, which were equivalent to the expected size; after the PCR product was purified and recovered, it was connected to the pGEM-T vector for cloning, transformation, sequencing, and sequence analysis It shows that the sizes of the cloned target DNA fragments are 470bp and 298bp respectively, which are the target DNA fragments expected to be amplified, which proves that the samples to be tested contain both PCV2 and PRV.

It can be seen from the above that the GC content of the two pairs of specific primers for the composite PCR of the present invention is equivalent, the cross-reaction between the primers is weak, and the annealing temperature is about 56 ° C, and the expected target fragment can be amplified with strong specificity; the amplified PCV2 and The size of PRV fragments is appropriate, and can be clearly distinguished in agarose gel electrophoresis, which is convenient for visually observing the experimental results; MgCl ₂ , dNTPs, rTaqDNApolymerase and primer concentrations are all comprehensively considered in terms of amplification efficiency, sensitivity, specificity, stability, etc. Factor design, can effectively detect PCV2 and PRV.

Example 2

Extract respectively porcine parvovirus (PPV), porcine escherichia coli (ETEC), porcine Actinobacillus pleuropneumoniae (APP) genomic DNA with viral genome DNA kit, and use the composite PCR primers and methods described in Example 1 to amplify , Electrophoresis to detect the specificity of the multiplex PCR.

The detection results are shown in Figure 2. The mixed DNA of PCV2 and PRV can amplify specific fragments, while PPV, ETEC, and APP do not amplify fragments, thus confirming the specificity of multiplex PCR amplification.

Example 3

Use an ultraviolet spectrophotometer to measure the concentration of the mixed template of PCV2DNA and PRV DNA. The concentration of the mixed DNA template is about 1 μg/μL. The mixed template is diluted 1:10 with TE buffer, and 2 μl of each dilution is used as a template. Compound PCR primers and methods described in Example 1 were used for compound PCR amplification and electrophoresis to detect the sensitivity of compound PCR.

The test results are shown in Figure 3. Two bands of 470bp and 298bp can still be amplified when the dilution is 10 ^-3 , and only one band of 470bp can be amplified when the dilution is 10 ^-5 . It shows that composite PCR can detect 1ng of PRV and 0.01ng of PCV2.

Example 4

Store PCV2 and PRV positive disease materials at -70°C; mix two pairs of specific primers, mix enzyme reagents, MgCl ₂ and dNTPs, and store at -20°C. Every one month, re-extract DNA from PCV2 and PRV-positive disease materials stored at -70°C, take out various mixed reagents of the compound PCR reaction system stored at -20°C for PCR amplification, and test for a total of 3 months to verify Stability of multiplex PCR.

The test results of 3 months showed that the expected target fragments could be amplified every time, and the visual concentration was equivalent, which proved the stability of the multiplex PCR reaction system.

Example 5

The detection of 73 clinical suspected disease materials in different regions of Zhejiang Province showed that this method not only greatly saves detection time but also consumes less reagents and reduces the chance of pollution. It has good practical application value and is suitable for popularization and application. The positive detection rate of PCV2 in the test is high, reaching 57.5%, which is consistent with the actual infection rate of PCV2; in addition, the positive detection rate of PCV2 and PRV mixed infection is 6.8%, which is consistent with the actual mixed infection rate. Thus, it can be seen that the present invention can effectively screen out infected individuals in pig herds, thereby preventing the spread of virus infection in time, and improving the virus prevention and control of PCV2 and PRV.

The above are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the examples shown herein, and all technical solutions under the idea of the present invention belong to the scope of protection of the present invention. It should be pointed out that for those skilled in the art, some modifications and embellishments without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Hangzhou Normal University

<120> A Primer Sequence and Method for Simultaneously Detecting PCV2 and PRV Infection

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 19

<212>DNA

<213> Artificial sequence

<400> 1

tccgcggggct ggctgaact 19

<210> 2

<211> 22

<212>DNA

<213> Artificial sequence

<400> 2

gggggaaagg gtgacgaact gg 22

<210> 3

<211> 18

<212>DNA

<213> Artificial sequence

<400> 3

tcctgtacgc cctattca 18

<210> 4

<211> 20

<212>DNA

<213> Artificial sequence

<400> 4

catccctggc tctagttatg 20

<210> 5

<211> 945

<212>DNA

<213> Porcine circovirus type 2

<400> 5

atgcccagca agaagaatgg aagaagcgga ccccaaccac acaaaaggtg ggtgttcacg 60

ctgaataatc cttccgaaga cgagcgcaag aaaatacggg agcttccaat ctcccttttt 120

gattatttta ttgttggcga ggagggtaat gaggaaggac gaacaccccca cctccagggg 180

ttcgctaatt ttgtgaagaa gcaaacattt aataaagtga aatggtattt cggtgcccgc 240

tgccacatcg agaaagcgaa aggaactgat cagcagaata aagaatactg cagtaaagaa 300

ggcaacttac tgatggaatg tggagctcct agatctcaag gacaacggag tgacctgtct 360

actgctgtga gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccct 420

gtaacgtttg tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatg 480

cagaagcgtg attggaagac caatgtacac gtcattgtgg ggccacctgg gtgtggtaaa 540

agcaaatggg ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaac 600

aagtggtggg acggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctgg 660

ctgccgtggg atgatctact gagattgtgt gatcgatatc cattgactgt agagactaaa 720

ggtggaactg tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttg 780

gaatggtact cctcaactgc tgtcccagct gtagaagctc tctatcggag gattacttcc 840

ttggtatttt ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcacc 900

ctttcccccc catgccctga atttccatat gaaataaatt actga 945

<210> 6

<211> 3422

<212>DNA

<213> Porcine pseudorabies virus

<400> 6

ggggccctcg ccgcgcgcct gcgccgcggc cgtggcggcg caggcgcgcg gcatggaggt 60

gacggagtcc gcgtacggcg accacatccg gcagtgcgtg tgcgccttca cgtcggagat 120

gggggtgtga ccctcgcccc tcccacccgc gccgcggccg gatggagacc gcgacggagg 180

caacgacgac ggcgtggggag ggggctcggg gcgcgtataa agccatgtgt atgtcatccc 240

aataaagttt gccgtgcccg tcaccatgcc cgcgtcgtcc gtgcgcctcc cgctgcgcct 300

cctgaccctc gcgggcctcc tggccctcgc gggggccgcc gccctcgccc gcggcgcgcc 360

gcagggtggg ccgccctcgc cgcagggggg tcccgcgccc accgcggcgc ccgcgcgcgg 420

gcccaccctg ttcgtcctgg tcggcgacgg ctccgcgggg ttcgtcttcc agctcggcgg 480

gctgggggcg ctcaacgaca cgcgcatccg cgggcacctg ctcggccggt acctcgtctc 540

gtaccaggtg gtgcccccgc ccgtctccgc gtggtacttt gtgcagcgcc cgcgcgagcg 600

cccgcgcctc tcggggccgc cctcgggcgc ggagctcgtg gccttcgacg cgcccggcgt 660

ccggcgcacg tacaccacgg cggcggtgtg gcccgcggag gtggccgtcc tcgcggacgc 720

ggaggcgcgc tgccccgcgg ccgtcttcaa cgtgacgctg ggcgaggcct tcctcggcct 780

gcgcgtcgcg ctgcgctcct tcctgccgct ggaggtcatc atctccgccg agcggatgcg 840

catgatcgcg cccccggcgc tcggcgcggg cctggagccg ccgggcccgc ccgcgggccg 900

cttccacgtg tacacgctcg gcttcctctc cgacggggcc atgcaccaga cgatgcgcga 960

cgtggccgcc tacgtgcacg agagcgacga ctacctcgcc cagctgtcgg cggcgcacgc 1020

ggccgccctg gccgccgtgg tgcagcccgg gccgtactac ttttaccgcg cggcggtgcg 1080

cctcggcgtg gccgccttcg tcttctccga ggcggcgcgc cgcgaccggc gcgcctcggc 1140

gccggcgctc ctgcgcgtcg agagcgacgc gcgcctgctc tcgcgcctgc tcatgcgcgc 1200

ggccggctgc cccgcgggct tcgccgggct cttcgacggg cgcgccgagc gcgtcccggt 1260

ggcgcccgcg gaccagctcc gcgccgcctg gaccttcggc gaggacccgg cgccccggct 1320

ggacctcgcg cgggcgaccg tcgccgaggc gtaccgccgc tccgtgcggg ggaagccctt 1380

cgaccagcag gcgctcttct tcgccgtcgc cctgctgctg cgcgccggcg gccccggcga 1440

cgcgcgcgag accctgctcc gcaccacggc catgtgcacc gcggagcgcg ccgccgcggc 1500

cgccgagctc acgcgggccg cgctctcgcc ggcggccgcg tggaacgagc ccttcagcct 1560

gctcgacgtc ctctcgccgt gcgccgtctc gctgcgccgc gacctcggcg gggacgccac 1620

cctggccaac ctgggcgccg cggcgcggct cgcgctggcg cccgccgggg ccccgggcgc 1680

ggcggcggcg acggacgagg gggcggagga ggaggaggag gaccccgtcg cgcgcgccgc 1740

gcccgagatc cccgccgagg cgctgctcgc cctgcccctg cgcgggggcg ccagcttcgt 1800

gttcacgcgc cggcgcccgg actgcggccc ggcgtacacg ctcggcggcg tggacatcgc 1860

caacccgctc gtgctcgccc tcgtcagcaa cgacagcgcc gcgtgcgact acacggaccg 1920

catgcccgag tcccagcacc tgccggcgac ggacaacccg tccgtgtgcg tgtactgcga 1980

ctgcgtgttc gtgcgctact cctccgcggg cacgatcctg gagaccgtcc tcatcgagtc 2040

caaggacatg gaggagcagc tcatggccgg cgccaactcc accatcccca gcttcaaccc 2100

gacgctgcac ggcggcgacg tcaaggccct gatgctcttc cccaacggca ccgtggtcga 2160

cctgctgtcg ttcacgtcga cgcggctcgc gcccgtgtcc ccggcctacg tggtggcctc 2220

cgtcgtgggg gcggccatca ccgtggggat cctgtacgcc ctattcaaga tgctctgcag 2280

cttctcctcc gagggctatt ctcggttaat aaacgccagg tcgtgaggcc cgcgccgggc 2340

cgcgaaccca gactctctgc gtgcgcgtgtttttccttgt cgggcgcggg gggagacgga 2400

ggggagacgg gagggggggg gaagacggca cgggcgccgt ccgtcgggga gacggcggga 2460

tgacatcacg agagtcgggt ggaggggatg tggggagcgc catccacggg ggaaggctgc 2520

tggatgacat aactagagcc agggatgagg atcctgctgg atgacataac tagagccagg 2580

gatgaggatc ccctttcccc cgttccccga gtatgcacca ctctctcccc gcaacacattt 2640

cccaccgggc gcctaacgtg gattgcaccc cggtccgcgc tacccggttg tgtgtgaggc 2700

ggacggagag ccgccctcgc cccggtcccc ccaaactcct gcggatgtgt ggtccgacga 2760

agcctccgcg gatgtgtccg ccgtaccctc gtccccctct ctcccacagt cctccctctc 2820

ccacagtcct ccctctccca cagtcctccc tctcccacag tcctccctct cccacagtcc 2880

tccctctccc acagtcctcc ctctcccaca gtcctccctc tccccacagtc ctccctctcc 2940

cacagtcctc cctctcccac agtcctccct ctcccacagt cctccctctc ccacagtcct 3000

ccctctccca cagtcctccc tctcccacag tcctccctcg tcccgaccgc cgaccgcact 3060

caagaacatt ttcacccgcg ctgaagaagg ggtctctaag gggtctctaa ggggtctcta 3120

aggggtctct aaggggtctc taaggggtct ctaaggggga ccaaagagta aagaccagag 3180

acgcatttta caatttcgtt gtacccgggt ttattgaaaa ctttgttcaa gagttaaagt 3240

gtctgccgct cgccggtgcg ccgcgccgag gccgcgatcc ggccgccgtg gaccgggggc 3300

ccgcgggctg gattcgacgg gacggcgggt ccgagcgacg gcggtccggc ggcgtttcgg 3360

ctcgcggtcc tcgcggcggc gacgatccgg cggtccacg acgatcgtgg tccgggggat 3420

cc 3422

Claims (4)

1. A primer sequence for simultaneous detection of PCV2 and PRV infection, characterized in that: comprise a PCV2 viral primer sequence and a PRV viral primer sequence; wherein, said PCV2 viral primer sequence comprises upstream primer PCV2-F1 and downstream primer PCV2-F2; The PRV virus primer sequence includes upstream primer PRV-F3 and downstream primer PRV-F4; the nucleotide sequences of the PCV2-F1, PCV2-F2, PRV-F3 and PRV-F4 are respectively as shown in the sequence table SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 shown. 2. a method for synchronously detecting PCV2 and PRV infection, is characterized in that, comprises the following steps: (1) design two pairs of specific primers as shown in claim 1 according to the nucleotide sequence design of PCV2ORF2 gene and PRV gH gene registered in GenBank; (2) Use the two pairs of specific primers to carry out compound PCR amplification on the sample to be tested to obtain the PCR product, which is detected by agarose gel electrophoresis; if there is a band of 470bp size, the sample to be tested contains PCV2 virus; if If a band with a size of 298bp appears, the sample to be tested contains PRV virus. 3. A method for synchronous detection of PCV2 and PRV infection according to claim 2, characterized in that: the reaction system of the multiplex PCR amplification is 50.0 μL, wherein: 10×Buffer 5.0 μL, _2.0mM MgCl 4.0 μL, 200 μM dNTPs 5.0 μL, 0.1 μM PCV2-F11.0 μL, 0.1 μM PCV2-F2 1.0 μL, 0.2 μM PRV-F3 2.0 μL, 0.2 μM PRV-F4 2.0 μL, rTaq DNApolymerase 0.5 μL, DNA template 2.0 μL, ddH ₂ O 27.5 μL. 4. a kind of method for simultaneously detecting PCV2 and PRV infection according to claim 2, is characterized in that: the reaction program of described compound PCR amplification is 95 ℃ 5min; 94 ℃ 30s, 56 ℃ 30s, 72 ℃ 45s, 35 cycles; final extension at 72°C for 10 min.