CN107227380A - The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection - Google Patents
The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection Download PDFInfo
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Abstract
The invention discloses the two pairs of specific primers and composite PCR detecting method synchronously detected for PCV2 and PRV, wherein two pairs specific primer G/C contents are suitable, annealing temperature is close, high specificity, primer dimer will not be produced, and PCV2 the and PRV clip sizes amplified are appropriate, can clearly be distinguished in agarose gel electrophoresis, be easy to intuitively observation experiment result.The present invention is by the reasonable setting of each technical parameter of composite PCR, synchronous detection PCV2 and PRV, simple, convenient, can formulate, technical requirements are low, with low cost, time saving and energy saving, and good specificity, sensitivity and stability are ensure that, suitable for clinical application.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of to be used for drawing for synchronous detection PCV2 and PRV mixed infections
Thing sequence and method.
Background technology
Porcine circovirus 2 type (porcine circovirus type 2, PCV2) and PRV (porcinep
Seudorabies virus, PRV) infection is generally existing and the serious DNA virus disease of harm in current China's Swine Production.
Wherein, the death rate is up to more than 50% when PCV2 breaks out, therefore it turns into most important infectious disease in current China's pig industry.
PRV belongs to herpetoviridae Alphaherpesvirinae, and it, which infects, can cause the miscarriage of adult in-pig, stillborn foetus, mummy, new life
Piglet performance nervous symptoms and high mortality within piglet acute death and three week old；Current China's scale pig farm according to investigations
The seroprevalence of pseudoabies is up to more than 70%, and harm is serious.2 kinds of cause of diseases of the above are clinically relatively conventional, and usually
Mixed infection, its disease triggered causes serious economic loss to pig industry.
The detection method that traditional PCV2 infection and PRV infect has virus purification, experimental animal inoculation and serological test
Etc. method, these methods are cumbersome, detection cycle length, the presence nonspecific reaction having and the low shortcoming of susceptibility,
The diseases prevention demand of aquaculture can not be met.With the development of molecular biology, PCR detection method can be fast and efficiently to disease
Substance is detected.
Polymerase chain reaction (PCR) is a kind of method of external enzyme' s catalysis specific DNA fragment, by high-temperature denatured, low temperature
Several steps reaction composition a cycles such as annealing (renaturation) and thermophilic extension, circulation progress enables target DNA to expand rapidly, had
There is the features such as high specificity, sensitivity are high, easy to operate, detection cycle is short, cannot be only used for Gene Isolation, clone and nucleic acid sequence
The basic research such as row analysis, it may also be used for the diagnosis of disease.
Standard PCR is simple to operate, and technical requirements are low, but needs to carry out multiple PCR detections for a variety of virus mixed infections,
And then detection cycle is extended, add testing cost.Composite PCR refers to add multipair primer in same PCR reaction systems,
The method that multiple target gene are expanded simultaneously；Using this method simultaneously detect the time saving and energy saving province of multiple target gene into
This, but primer present in reaction system is readily formed the primer dimer of complexity, and then influence sensitivity and the standard of detection
True property, thus composite PCR successful key be design primer specificity, reaction system and program optimization.
Have the method detected by multiple RT-PCR to a variety of viruses of pig in the prior art, but its to RNA concentration,
Quality requirement is higher, and the pollution of genomic DNA and RNase is highly prone in detection process, therefore, and it will to operating personnel's technology
Ask higher, be unfavorable for wide popularization and application.In addition, there is also the detection method of multiple fluorescence PCR, but its probe marks difference
It is easy to interfere with each other between the fluorophor of emission wavelength, and then influences specific detection.
As can be seen here, very big development space, ability are still suffered from for the method that PCV2 and PRV infection synchronizes detection
Field technique personnel urgently design the PCV2 that a kind of easy to operate, technical requirements are low, specific high, stability is strong, reproducible,
PRV synchronization detecting methods.
The content of the invention
Based in place of the deficiencies in the prior art, present invention offer is a kind of can to detect PCV2 and PRV mixed infections simultaneously
Method.
The present invention is adopted the technical scheme that to achieve these goals：
A kind of primer sequence of synchronous detection PCV2 and PRV infection, it is characterised in that：Including the viral primer sequences of PCV2 and
PRV virus primer sequences；Wherein, the viral primer sequences of the PCV2 include sense primer PCV2-F1 and anti-sense primer PCV2-
F2；The viral primer sequences of the PRV include sense primer PRV-F3 and anti-sense primer PRV-F4；The PCV2-F1, PCV2-F2,
PRV-F3 and PRV-F4 nucleotide sequence respectively as sequence table SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and
Shown in SEQ ID NO.4.
A kind of method of synchronous detection PCV2 and PRV infection, it is characterised in that comprise the following steps：
(1) according to the nucleotide sequence of the PCV2ORF2 genes and PRV gH genes logged in GenBank design such as right
It is required that two pairs of specific primers shown in 1；
(2) composite PCR amplification is carried out to sample to be tested using two pairs of specific primers, obtains PCR primer, carry out fine jade
Sepharose electrophoresis detection；If occurring in the band of 470bp sizes, testing sample containing PCV2 viruses；If it is big 298bp occur
Small band, then contain PRV viruses in testing sample.
Further, the reaction system of the composite PCR amplification is 50.0 μ L, wherein：10×Buffer 5.0μL、
2.0mM MgCl24.0μL、200μM dNTPs 5.0μL、0.1μM PCV2-F11.0μL、0.1μM PCV2-F21.0μL、0.2μ
M PRV-F32.0 μ L, 0.2 μM of PRV-F42.0 μ L, the μ L of rTaq DNA polymerase 0.5, DNA profiling 2.0 μ L, ddH2O
27.5μL。
Further, the response procedures of the composite PCR amplification are 95 DEG C of 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 45s,
35 circulations；Last 72 DEG C of extensions 10min.
Beneficial effect：The invention provides for PCV2 and PRV synchronously two pairs of specific primers of detection and composite PCR inspections
Survey method.By compare in GenBank databases 15 kinds of PCV2 Strain, the gene common characteristic of 18 kinds of PRV Strain and
PCV2 ORF1, PRV gH genes nucleotide sequence, have separately designed two pairs of specificity using Primer Premier softwares and have drawn
Thing, quite, annealing temperature is close, high specificity for its G/C content, will not produce primer dimer, and the PCV2 amplified and
PRV clip sizes are appropriate, can clearly be distinguished in agarose gel electrophoresis, be easy to intuitively observation experiment result.This hair
The bright reasonable setting by each technical parameter of composite PCR, synchronous detection PCV2 and PRV is simple, convenient, can formulate, skill
Art requires low, with low cost, time saving and energy saving, and ensure that good specificity, sensitivity and stability, suitable for clinical expansion
Using.
Brief description of the drawings
Fig. 1 show composite PCR amplification result of the test of the present invention；M is pUC19 DNA/Msp Ι Marker in figure, and 1 is
PCV2 DNA cloning products；2 be PRV DNA cloning products；PCV2 DNA and PRV DNA hybrid templates amplified production.
Fig. 2 show the specific test result of composite PCR of the present invention；M is pUC19 DNA/Msp Ι Marker, 1 in figure
For PCV2 DNA and the amplified production of PRV DNA hybrid templates；2 be PPV DNA cloning products；3 be ETEC DNA cloning products；
4 be APP DNA cloning products.
Fig. 3 show the sensitivity tests result of composite PCR of the present invention；M is pUC19 DNA/Msp Ι Marker in figure,
1-8 is PCV2 DNA and PRV DNA hybrid templates 10-1-10-8Amplified production under extension rate.
Embodiment
The present invention is described in further detail with reference to embodiment：
Embodiment 1
(1) design and synthesis of primer
By compare in GenBank databases 15 kinds of PCV2 Strain, the gene common characteristic of 18 kinds of PRV Strain and
PCV2 ORF1 (SEQ ID NO.5), PRV gH (SEQ ID NO.6) gene nucleotide sequence, using Primer Premier
Software has separately designed two pairs of specific primers, including the viral primers of PCV2 and the viral primers of PRV.
Wherein, the viral primers of PCV2 include sense primer PCV2-F1 and anti-sense primer PCV2-F2；PRV virus primers include
Sense primer PRV-F3 and anti-sense primer PRV-F4；PCV2-F1, PCV2-F2, PRV-F3 and PRV-F4 nucleotide sequence difference
As shown in table 1.Primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd, it is contemplated that amplified production size be respectively 470bp and
298bp。
Table 1
(2) extraction of virus genom DNA
Positive each 0.1 gram of the pathological material of disease of PCV2, PRV is taken respectively, is placed in Eppendorf pipes, TE buffer solution 1ml is added, with sth. made by twisting
Frotton is twisted homogenate, -70 DEG C of freeze thawing three times, centrifuging and taking supernatant 0.2ml, then extracts viral DNA with viral DNA extracts kit,
Finally elute in 50 μ l TE buffer solutions, in -20 DEG C of preservations.
(3) composite PCR is detected；
With PCV2DNA or PRV DNA or PCV2DNA and PRV DNA 1：1 mixing carries out composite PCR expansion as DNA profiling
Increase, the reaction system of composite PCR amplification is 50.0 μ L, wherein：10×Buffer 5.0μL、2.0mM MgCl24.0μL、200μM
dNTPs 5.0μL、0.1μM PCV2-F11.0μL、0.1μM PCV2-F21.0μL、0.2μM PRV-F32.0μL、0.2μM
PRV-F42.0 μ L, the μ L of rTaq DNA polymerase 0.5, DNA profiling 2.0 μ L, ddH2O 27.5μL。
The response procedures of composite PCR amplification are 95 DEG C of 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 45s, 35 circulations；Finally
72 DEG C of extension 10min, 4 DEG C save backup.
After reaction terminates, row agarose gel electrophoresis detection is entered to pcr amplification product, testing result is as shown in Figure 1.No. 1
A clearly purpose band, about 470bp, with expected sizableness are amplified in swimming lane；After PCR primer purifying is reclaimed, even
It is connected in pGEM-T carriers and clones, converts, being sequenced, sequence analysis shows, the target DNA fragment size cloned is 470bp, is
It is expected that the target DNA fragment of amplification, it was demonstrated that contain PCV2 in sample.
A clearly purpose band, about 298bp, with expected sizableness are amplified in No. 2 swimming lanes；PCR primer is pure
Change after reclaiming, be connected in pGEM-T carriers and clone, convert, being sequenced, sequence analysis shows that the target DNA fragment cloned is big
Small is 298bp, for the target DNA fragment of expected amplification, it was demonstrated that contain PRV in testing sample.
Two clearly purpose bands are amplified simultaneously in No. 3 swimming lanes, 470bp and 298bp is respectively may be about, with expected size
Quite；After PCR primer purifying is reclaimed, it is connected in pGEM-T carriers and clones, converts, being sequenced, sequence analysis shows, is cloned
Target DNA fragment size be respectively 470bp and 298bp, for the target DNA fragment of expected amplification, it was demonstrated that same in testing sample
Shi Hanyou PCV2 and PRV.
From the foregoing, it will be observed that composite PCR two of the present invention is suitable to specific primer G/C content, cross reaction is weak between primer, annealing
Temperature at 56 DEG C or so, it is amplifiable go out expected purpose fragment, high specificity；PCV2 the and PRV clip sizes amplified are fitted
When can clearly be distinguished in agarose gel electrophoresis, be easy to intuitively observation experiment result；MgCl2、dNTPs、rTaq
DNApolymerase and primer concentration are to consider many factors such as amplification efficiency, sensitivity, specificity, stability to set
Meter, it can effectively detect PCV2 and PRV.
Embodiment 2
Extract pig parvoviral (PPV), swine escherichia coli (ETEC), pig pleura respectively with virus genom DNA kit
Actinobacillus (APP) genomic DNA, composite PCR primer and method described in Application Example 1 are expanded, electrophoresis,
To detect the specificity of composite PCR.
Testing result is as shown in Fig. 2 PCV2 and PRV hybrid dna can amplify specific fragment, and PPV, ETEC, APP
Do not amplify fragment, thereby confirm the specificity of composite PCR amplification.
Embodiment 3
The concentration of PCV2DNA and PRV DNA hybrid templates is determined with ultraviolet specrophotometer, hybrid dna template concentrations are about
For 1 μ g/ μ L, hybrid template is made 1 with TE buffer solutions:10 are serially diluted, and each dilution factor takes 2 μ l as template, application implementation
Composite PCR primer and method described in example 1 carry out composite PCR amplification, electrophoresis, to detect the sensitiveness of composite PCR.
Testing result is as shown in figure 3,10-3Remain to amplify 470bp and the bands of 298bp two, 10 during extension rate-5Dilution
When be only capable of amplifying the bands of 470bp mono-.Illustrate that composite PCR can detect 1ng PRV and 0.01ng PCV2.
Embodiment 4
By the positive pathological material of diseases of PCV2 and PRV in -70 DEG C of preservations；Two pairs of specific primer mixing, enzymatic reagent, MgCl2And dNTPs
Mixing, in -20 DEG C of preservations.Every 1 month, the positive pathological material of disease of the PCV2 and PRV of -70 DEG C of preservations is extracted into DNA again, -20 are taken out
DEG C preserve composite PCR reaction system various mix reagents enter performing PCR amplification, altogether detect 3 months, to verify composite PCR
Stability.
The testing result of 3 months is shown：Expected purpose fragment can be amplified every time, and it is suitable to observe concentration, it was demonstrated that
The stability of composite PCR reaction system.
Embodiment 5
73 parts of doubtful pathological material of diseases of clinic of Zhejiang Province different regions are detected, as a result show that this method is not only greatlyd save
Detection time and consumption reagent it is few, reduce opportunities for contamination, with good actual application value, suitable for popularization and application.Detection
Middle PCV2 positive rate is high, reaches 57.5%, the positive rate and PCV2 actual infection rate matching degree are higher；Separately
Outside, PCV2 and PRV mixed infections positive rate are 6.8%, are consistent with actual mixed infection rate；As can be seen here, beneficial to this
Invention can the infected individuals that go out in swinery of Effective selection, and then the diffusion for preventing virus from infecting in time improves PCV2 and PRV disease
Malicious prevention and control dynamics.
It the above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited to reality shown in this article
Example is applied, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that being led for this technology
For the those of ordinary skill in domain, some modifications and retouching without departing from the principles of the present invention also should be regarded as the present invention's
Protection domain.
SEQUENCE LISTING
<110>Hangzhou Pedagogic University
<120>The primer sequence and method of a kind of synchronous detection PCV2 and PRV infection
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
tccgcgggct 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 2 type
<400> 5
atgcccagca agaagaatgg aagaagcgga ccccaaccac acaaaaggtg ggtgttcacg 60
ctgaataatc cttccgaaga cgagcgcaag aaaatacggg agcttccaat ctcccttttt 120
gattatttta ttgttggcga ggagggtaat gaggaaggac gaacacccca 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>PRV
<400> 6
ggggccctcg ccgcgcgcct gcgccgcggc cgtggcggcg caggcgcgcg gcatggaggt 60
gacggagtcc gcgtacggcg accacatccg gcagtgcgtg tgcgccttca cgtcggagat 120
gggggtgtga ccctcgcccc tcccacccgc gccgcggccg gatggagacc gcgacggagg 180
caacgacgac ggcgtgggag 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 gtgcgcgtgt ttttccttgt 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 gcacacattt 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 tcccacagtc 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 cggtcccacg acgatcgtgg tccgggggat 3420
cc 3422
Claims (4)
1. a kind of primer sequence of synchronous detection PCV2 and PRV infection, it is characterised in that：Including the viral primer sequences of PCV2 and
PRV virus primer sequences；Wherein, the viral primer sequences of the PCV2 include sense primer PCV2-F1 and anti-sense primer PCV2-
F2；The viral primer sequences of the PRV include sense primer PRV-F3 and anti-sense primer PRV-F4；The PCV2-F1, PCV2-F2,
PRV-F3 and PRV-F4 nucleotide sequence respectively as sequence table SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and
Shown in SEQ ID NO.4.
2. a kind of method of synchronous detection PCV2 and PRV infection, it is characterised in that comprise the following steps：
(1) according to the nucleotide sequence of the PCV2ORF2 genes and PRV gH genes logged in GenBank design such as claim 1
Two pairs of shown specific primers；
(2) composite PCR amplification is carried out to sample to be tested using two pairs of specific primers, obtains PCR primer, carry out agarose
Detected through gel electrophoresis；If occurring in the band of 470bp sizes, testing sample containing PCV2 viruses；If there are 298bp sizes
Band, then contain PRV viruses in testing sample.
3. a kind of method of synchronous detection PCV2 and PRV infection according to claim 2, it is characterised in that：It is described compound
The reaction system of PCR amplifications is 50.0 μ L, wherein：10×Buffer 5.0μL、2.0mM MgCl24.0μL、200μM dNTPs
5.0μL、0.1μM PCV2-F11.0μL、0.1μM PCV2-F2 1.0μL、0.2μM PRV-F32.0μL、0.2μM PRV-
F42.0 μ L, the μ L of rTaq DNApolymerase 0.5, DNA profiling 2.0 μ L, ddH2O 27.5μL。
4. a kind of method of synchronous detection PCV2 and PRV infection according to claim 2, it is characterised in that：It is described compound
The response procedures of PCR amplifications are 95 DEG C of 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 45s, 35 circulations；Last 72 DEG C of extensions
10min。
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