CN102373299A - PCV2 (Porcine Circovirus 2), PRV (Pseudorabies Virus) and SIV (Swine Influenza Virus) H9 subtype multiplex SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) primer and detection method thereof - Google Patents

Abstract

The invention discloses a PCV2 (Porcine Circovirus 2), PRV (Pseudorabies Virus) and SIV (Swine Influenza Virus) H9 subtype multiplex SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) primer and a detection method thereof. The sequences of SIV H9 subtype primers are as follows: an upstream primer P1: 5'-GGAACAACGCTTACCCTG-3', and a downstream primer P2: 5'-GAGACCATTGACAAGAGGC-3'; the sequences of PRV primers are as follows: an upstream primer P3: 5'-GGCATCGGCGACTACCT-3', and a downstream primer P4: 5'-CGTCGTGCAGCGTGTAGAG-3'; and the sequences of PCV2 primers are as follows: an upstream primer P5: 5'-GGGCCAGAATTCAACCTTACCC-3', and a downstream primer P6: 5'-CGCACCTTCGGATATACTGTCA-3'. In the invention, specific primers for amplifying SIV H9 subtype, PCV2 and PRV are designed respectively, the SIV H9 subtype, PCV2 and PRV can be detected simultaneously with a triple SYBR Green I real-time quantitative PCR detection method, and the simultaneously-detected extreme copy numbers of the SIV H9 subtype, PCV2 and PRV are 184 copies/mu L, 207 copies/mu L and 193 copies/mu L respectively. The specific test result is good, and only three specific peak values appear; and a repetitive test has high stability and the maximum advantages of detecting three kinds of DNA (Deoxyribose Nucleic Acid) viruses, i.e., SIV H9 subtype, PCV2 and PRV simultaneously and contributes to identifying pregnant swine reproductive failure viruses.

Description

PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer and detection method thereof

Technical field

The present invention relates to a kind of detection method of virus, be specifically related to PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer and detection method thereof.

Background technology

PRV (PRV) and porcine circovirus 2 type (PCV2) can both cause gestation to cause that boar is sterile in various degree, i.e. boar testis swelling, and semen quality descends, and loses the ability of kind using; Sow shows as out of heat, returns feelings, and is infertile; Pregnant sow is miscarried, premature labor, stillborn foetus, mummy, weak son and newborn piglet morbidity and cause mass mortality etc., has caused enormous economic loss to pig industry.Effective control to this type transmissible disease is to guarantee one of pig industry key of healthy development factor.In the animal influenza, porcine influenza is that economic implications is maximum except that bird flu, and public health meaning particularly important.The porcine influenza epidemic situation of Mexico's outburst in 2009 causes hundreds of people dead, and people's parainfluenza that porcine influenza causes spreads rapidly in the world.So the prevention of porcine influenza and control substantial connection are to human public health security.

At present, have much to the diagnostic method of above three kinds of virus diseases, but shortcoming such as these methods have complicated operation, waste time and energy, susceptibility difference and negative rate height; Though clinical already used PCR diagnostic techniques has highly sensitive, high specificity, advantage such as quick, easy, can not be accurately quantitative; And the TaqMan fluorescence quantifying PCR method can be quantitative, but cost is higher, and need synthetic specific probe.Need low, easy, quick, special, the quantitative detection method of research and development cost badly.Clinically; These several kinds viruses usually occur with the form of polyinfection; Lean on isolated viral and antibody test to make a definite diagnosis and cause false negative easily, and complex operation, higher to the operational requirement of instrument; Required time is long, need badly a kind of simple to operate, detect fast, differential diagnosis method that accuracy rate is high.

Summary of the invention

The technical problem that the present invention will solve is that PCV2, PRV and these several kinds viruses of SIV H9 hypotype usually occur with the form of polyinfection; Lean on isolated viral and antibody test to make a definite diagnosis and cause false negative easily; And complex operation, a kind of PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer and detection method thereof are provided.

Technical scheme of the present invention is: PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer:

The sequence of SIV H9 hypotype primer is following:

Upstream primer P1:5 '-GGAACAACGCTTACCCTG-3 ';

Downstream primer P2:5 '-GAGACCATTGACAAGAGGC-3 ';

The sequence of PRV primer is following:

Upstream primer P3:5 '-GGCATCGGCGACTACCT-3 ';

Downstream primer P4:5 ' CGTCGTGCAGCGTGTAGAG-3 ';

The sequence of PCV2 primer is following:

Upstream primer P5:5 '-GGGCCAGAATTCAACCTTACCC-3 ';

Downstream primer P6:5 '-CGCACCTTCGGATATACTGTCA-3 '.

Said primer detects the multiple SYBR Green I real-time fluorescence PCR detection method of PCV2, PRV and SIV H9 hypotype; After comprising the DNA that extracts virus; By following SYBR Green I real-time fluorescence PCR reaction system and reaction conditions virus is detected; Said SYBR Green I real-time fluorescence PCR reaction adds following composition in 25 μ l systems:

Said reaction conditions is: 95 ℃ of preparatory sex change 1min; Get into circulation, 95 ℃ of sex change 10s, 58 ℃ of annealing 15s, 72 ℃ are extended 15s; 40 circulations are warming up to 95 ℃ of 15s after the loop ends, reduce to 65 ℃ of 15s again; Begin to be incremented to 94 ℃, gather the solubility curve that fluorescent signal draws amplified production, finish reaction in 95 ℃ of 15s from 65 ℃.

The invention has the beneficial effects as follows: the present invention designs the Auele Specific Primer of amplification SIV H9 hypotype, PCV2 and PRV respectively, can detect SIV H9 hypotype, PCV2 and PRV simultaneously through triple SYBR Green I real-time quantitative PCR detection methods.The present invention utilizes the TM value to distinguish different nucleic acid fragments, and the TM value of nucleic acid fragment is main relevant with GC content, sequence length and structure.The long 209bp of amplified fragments gene order of SIV H9 hypotype, its TM value is about 92 ℃; The long 171bp of PCV2 amplified fragments, the TM value is below 85 ℃; The amplified fragments of PRV is 136bp, about 89 ℃ of TM values, and the TM value of SIV, PCV2 and PRV can well be distinguished.

In the present invention; The TM value of the TM value of SIV H9 hypotype amplified fragments, the TM value of PCV2 amplified fragments and PRV amplified fragments can be in certain TR change; The TM value variation range of SIV H9 hypotype is 91.8-92.0 ℃, and the TM value variation range of PCV2 is 84.6-84.9 ℃, and the TM value variation range of PRV is 89.2-89.6 ℃; The TM value of 3 kinds of viruses differs bigger, can utilize three specific peaks of melting curve that these 3 kinds of viruses are differentiated whereby.

Susceptibility of the present invention shows that the limit copy number that SIV H9 hypotype, PCV2 and PRV detect simultaneously is respectively 184 copy/μ L, 207 copy/μ L, 193 copy/μ L.Specificity test-results of the present invention is good; Three specific peak values only appear; The sharpest edges that replica test has good stability and this test are to detect simultaneously SIV H9 hypotype, PCV2 and 3 kinds of dna virus of PRV, help the discriminating of pregnant sow breeding difficulty venereal disease poison.

The present invention has set up detection SIV H9 hypotype, PCV2 and the multiple SYBR Green of PRV I fluorescence quantifying PCR method; The result shows; This method has specificity, repeatability and susceptibility preferably; For the detection of SIV H9 hypotype, PCV2 and PRV provides a kind of new method, can be used as the purification detection method of large-scale pig farm SIV H9 hypotype, PCV2 and PRV, set up the swinery of no SIV H9 hypotype, PCV2 and PRV; It simultaneously also is the Molecule Epidemiology Investigation that SIV H9 hypotype, PCV2 and PRV infect; Research SIV H9 hypotype, PCV2 and PRV molecular pathogenesis, the development and the exploitation of SIV H9 hypotype, PCV2 and PRV diagnostic kit, the immunologic mechanism of medicine or vaccine provides test basis and technique means.

Description of drawings

Fig. 1 is a SIV H9 hypotype portion gene plasmid PCR qualification result, M.DL 2000Marker, 1. negative control, 2. purpose fragment;

Fig. 2 is a PCV2 portion gene plasmid PCR qualification result, M.DL 2000Marker, 1. negative control, 2. purpose fragment;

Fig. 3 is a PRV portion gene plasmid PCR qualification result, M.DL 2000Marker, 1. negative control, 2. purpose fragment;

Fig. 4 is the analysis of composite S YBR Green I quantitative fluorescent PCR reaction melting curve;

Fig. 5 is the analysis of multiple SYBR Green I quantitative fluorescent PCR atopic test melting curve;

Fig. 6 is that the reaction of composite S YBR Green I quantitative fluorescent PCR is with the analysis of concentration replica test melting curve;

Fig. 7 is the analysis of different concns replica test melting curve;

Fig. 8 is the analysis of different concns replica test melting curve;

Fig. 9 is the analysis of different concns replica test melting curve.

Embodiment

PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer:

The sequence of SIV H9 hypotype primer is following:

Upstream primer P1:5 '-GGAACAACGCTTACCCTG-3 ';

Downstream primer P2:5 '-GAGACCATTGACAAGAGGC-3 ';

The sequence of PRV primer is following:

Upstream primer P3:5 '-GGCATCGGCGACTACCT-3 ';

Downstream primer P4:5 '-CGTCGTGCAGCGTGTAGAG-3 ';

The sequence of PCV2 primer is following:

Upstream primer P5:5 '-GGGCCAGAATTCAACCTTACCC-3 ';

Downstream primer P6:5 '-CGCACCTTCGGATATACTGTCA-3 '

Said primer detects the multiple SYBR Green I real-time fluorescence PCR detection method of PCV2, PRV and SIV H9 hypotype, comprise extract viral DNA after, by following SYBR Green I real-time fluorescence PCR reaction system and reaction conditions virus is detected:

Said SYBR Green I real-time fluorescence PCR reaction system adds following composition in 25 μ l systems:

Said reaction conditions is: 95 ℃ of preparatory sex change 1min; Get into circulation, 95 ℃ of sex change 10s, 58 ℃ of annealing 15s, 72 ℃ are extended 15s; 40 circulations are warming up to 95 ℃ of 15s after the loop ends, reduce to 65 ℃ of 15s again; Begin to be incremented to 94 ℃, gather the solubility curve that fluorescent signal draws amplified production, finish reaction in 95 ℃ of 15s from 65 ℃.

Concrete experimental implementation is following:

1 materials and methods

1.1 material

1.1.1 strain, cell strain and bacterial strain

PRV, PPV standard strain are available from China Veterinary Drugs Supervisory Inst.; CSFV, SIV H9 hypotype, PRRSV, PCV2 are available from Henan Province's animal food safety key lab; DH5 α competent cell is available from precious biotechnology ltd.

1.1.2 solution commonly used and substratum

The LB liquid nutrient medium, 4 ℃ of preservations; Penbritin (Ampicillin, Amp) stock solution, 100mg/mL; X-gal, concentration is 20mg/mL, preserves with brown bottle or with the bottle of aluminium foil parcel, IPTG liquid, concentration is 100mg/mL; Be stored in-20 ℃ subsequent use.

1.1.3 key instrument and reagent

The ultraviolet gel imaging system is available from U.S. ALPHA INNOTECH company; Agilent Mx3005P type real-time quantitative PCR amplification appearance available from U.S. Agilent Stratagene company, PTC-200 type PCR appearance available from U.S. MJ company etc.

SYBR

Premix Ex Taq ^TMII, EX TaqDNA polysaccharase, DNA marker DL2000 are all available from the precious biotechnology in Dalian ltd; Protein K is available from Huamei Bio-Engrg Co.; T4 dna ligase, PGEM-T Easy plasmid vector are all available from Promega company; Dna gel reclaims test kit available from liking to pursue progress Bioisystech Co., Ltd; Plasmid extraction kit is available from vast Tyke, Beijing biotech firm; RNA extracts test kit available from Shanghai JaRa Bioisystech Co., Ltd; The reverse transcription test kit is available from U.S. MBI company.

1.2 method

1.2.1 primer design is with synthetic

Announce PRV gH gene with GenBank; The SIVH9 gene is canonical sequence (EF055887); PCV2 ORF2 gene is canonical sequence (AF027217), designs a pair of Auele Specific Primer respectively with Primer Premier 6.0 biosoftwares, and the sequence of primer is as shown in table 1.

Table 1 fluorescence PCR primer sequence

1.2.2PCV2, the extraction of PRV, PPV DNA

Traditional Proteinase K process for extracting: get the tissue that contains these three kinds of viruses that had detected, shred, add an amount of PBS and grind, get the tissue juice after 450 μ L grind; Add equal-volume sample dissociation damping fluid [0.02mol/L Tris HCl (PH 7.5), 0.03mol/L EDTA, 1%SDS]; Mixing adds Proteinase K (final concentration is 200 μ g/m L), 55 ℃ of water-bath 1h again; Add isopyknic balance phenol then, mixing, the centrifugal 5min of 12000r/min.Get supernatant, through phenol: chloroform: primary isoamyl alcohol (25: 24: 1) once more after the extracting with the isopropanol precipitating of 2 times of volumes, place 30min for-20 ℃; The centrifugal 5min of 12000r/min, 70% washing with alcohol 2 times, drying; Add 25 μ L ultrapure waters dissolvings, be stored in-20 ℃ subsequent use.

1.2.3CSFV, the preparation of PRRSV, SIV H9 subtype cDNA

Get the tissue that contains these three kinds of viruses that had detected, shred, add an amount of PBS and grind, get the tissue juice after 450 μ L grind, press the test kit explanation then and extract and reverse transcription.

1.2.4 the preparation of plasmid template standard substance

PCV2DNA, PRV DNA and SIV H9 subtype cDNA to extract are template, with optimized conditions amplification gene fragment.In 50 μ L reaction systems, add successively respectively: EX TaqDNA polysaccharase 28 μ L, primer concentration P1/P3/P5, P2/P4/P6 are 0.5 μ m/L, template 2 μ L; Mend to 50 μ L with distilled water at last, the EP pipe is put the PCR appearance, increase by following program: behind 95 ℃ of preparatory sex change 5min; Advance the people 95 ℃ of 30s that circulate, 56 ℃ of 30s, 72 ℃ of 20s; After 30 circulations, 72 ℃ are extended 10min, and 4 ℃ are finished reaction.2.0% agarose gel electrophoresis.

To amplify the purpose fragment and reclaim the test kit recovery by dna gel; Connecting the test kit specification sheets according to carrier is connected the purpose fragment with pGEM-T Easy carrier; Transform the DH5a competent cell, select positive bacterium colony, carry out after bacterium liquid PCR identifies; Extract DNA with plasmid extraction kit explanation, send precious biotechnology ltd to check order.Through the positive plasmid of sequence verification as the template standard article, and called after pGEM-H9, pGEM-PRV and pGEM-PCV2 respectively.1.2.5SIV the optimization of H9 hypotype, PRV, PCV2 composite S YBR Green I quantitative fluorescent PCR reaction system condition

SYBR Green I quantitative fluorescent PCR reaction system is following:

In 25 μ L systems, add following composition:

Reaction conditions is: 95 ℃ of 1min; 95 ℃ of 10s, 58 ℃ of 15s, 72 ℃ of 15s carry out 40 circulations altogether, are warming up to 95 ℃ of 15s after the loop ends, reduce to 65 ℃ of 15s again, begin to be incremented to 94 ℃ from 65 ℃, gather the solubility curve that fluorescent signal draws amplified production, finish reaction in 95 ℃ of 15s.

1.2.5.1 the optimization of primer concentration

Primer concentration 0.3 μ L, 0.4 μ L, 0.5 μ L, 0.6 μ L, 0.7 μ L, 0.8 μ L, 0.9 μ L with 50pmol/ μ L carries out the quantitative fluorescent PCR reaction respectively, chooses best primer concentration amplicon virus.

1.2.5.2 the optimization of annealing temperature

Triple SYBR Green I quantitative fluorescent PCRs react with 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃ annealing temperature respectively, select best annealing temperature.

1.2.5.3SYBR

Premix Ex Taq ^TMThe optimization of II concentration

SYBR among the present invention

Premix Ex Taq ^TMThe concentration of II is 5U/ μ L, and reaction system SYBR Green I quantitative fluorescent PCR TV is fixing, through changing SYBR

Premix Ex Taq ^TMThe addition of II screens SYBR in the quantitative fluorescent PCR reaction system The optimum concn of Premix Ex Taq.Screen with the amount of 10 μ L, 11.5 μ L, 13 μ L, 14.5 μ L, 16 μ L, 17.5 μ L respectively.

1.2.6 specificity check

According to SYBR Green I fluorescent quantitation reaction system; Add SIV H9 subtype cDNA, PRV DNA, PCV2DNA (about 20ng) respectively; Pig parvoviral (PPV) DNA 1 μ L (about 20ng); Porcine reproductive and respiratory syndrome virus (PRRSV) cDNA 1 μ L (about 20ng) establishes negative control simultaneously, verifies its specificity.

1.2.7 repeatability check

The plasmid standard of choosing SIV H9 hypotype, PRV, the same concentration of PCV2 carries out composite S YBR Green I quantitative fluorescent PCR reaction replica test, reaction repeated three times; The plasmid standard of different concns is carried out composite S YBR Green I quantitative fluorescent PCR reaction replica test, reaction repeated three times; Through the TM value and the solubility curve analysis of every kind of virus, the stability of checking SIV H9 hypotype, PRV, the multiple SYBR Green of PCV2 I fluorescence quantifying PCR method.

1.2.8 quantitative fluorescent PCR sensitivity testing

Respectively the recombinant plasmid of SIVH9 hypotype, PRV, PCV2 is surveyed OD ₂₆₀Be worth, calculate the copy number of every microlitre, carry out 10 times of gradient dilutions then, and carry out the reaction of composite S YBR Green I quantitative fluorescent PCR, confirm the susceptibility of every kind of virus of composite S YBRGreen I quantitative fluorescent PCR reaction detection as template.

1.2.9 doubtful SIV H9 hypotype, PRV, PCV2 samples detect the comparison that reaches with conventional PCR detection method

Get the pathological material of disease that the doubtful SIV H9 hypotype of picking up from a plurality of districts and cities pig farm in the Henan Province, PRV, PCV2 infect and carry out composite S YBR Green I quantitative fluorescent PCR and detection, to verify the practicality of this method.

2 results

2.1 conventional plasmid pcr amplification

Shown in Fig. 1-3SIV H9 hypotype, PCV2, PRV portion gene plasmid PCR qualification result, amplified respectively and the fragment of estimating big or small corresponding to 209bp, 171bp and 136bp.PGEM-H9, pGEM-PCV2 and pGEM-PRV cut evaluation through EcoR I enzyme, and it is consistent with the band of expection to obtain product.Order-checking is compared with domestic popular strain, and nucleotide homology is 100%, 97.6%, more than 99.6%.

2.2 the calculating of plasmid concentration

The SIV H9 hypotype, PCV2, the PRV plasmid DNA concentration that record extraction are respectively: pGEM-H9 mass concentration=52.7 μ g/ μ L, pGEM-PCV2 mass concentration=19.9 μ g/ μ L, pGEM-PRV mass concentration=32.4 μ g/ μ L.Through calculating, the pGEM-H9 plasmid concentration is 2.96 * 10 ¹⁰Copy/mL, pGEM-PCV2 plasmid concentration are 2.19 * 10 ¹¹Copy/mL, pGEM-PRV plasmid concentration are 6.68 * 10 ¹⁰Copy/mL.

2.3SIV the optimum result of H9 hypotype, PCV2, the triple SYBR Green of PRV I quantitative fluorescent PCR reaction conditions

2.3.1 primer concentration optimum result

It is 25 μ L that SIV H9 hypotype, PCV2, PRV primer all adopt the quantitative response system of 50pmoL/ μ L 0.5 μ L, produces specific single peak value, and negative control does not have the specificity peak value and produces.The best primer concentration of SIV H9 hypotype, PCV2, the reaction of the triple SYBR Green of PRV I quantitative fluorescent PCR is 0.5 μ L50pmoL/ μ L.

2.3.2 annealing temperature optimum result

SIV H9 hypotype, PCV2, PRV have all produced specific peak value when 58 ℃ of annealing temperatures.SIV H9 hypotype TM value is respectively 91.7 ℃, and 91.8 ℃, 92.0 ℃.The PCV2TM value is respectively 84.8 ℃, and 84.9 ℃, 85.1 ℃; The PRVTM value is respectively 89.3 ℃, and 89.3 ℃, 89.5 ℃.Negative control does not produce the specificity peak value.The annealing temperature optimum result of SIV H9 hypotype, PCV2, the reaction of the triple SYBRGreen I of PRV quantitative fluorescent PCR is 58 ℃.

2.3.3SYBR

Premix Ex Taq ^TMThe optimum result of II concentration

SYBR in SIV H9 hypotype, PCV2, the reaction of the triple SYBR Green of PRV I quantitative fluorescent PCR

Premix ExTaq ^TMThe II addition is when 14.5 μ L, and the three all can produce specific peak value, and negative control does not then produce the specificity peak value.

2.4 confirming of composite S YBR Green I quantitative fluorescent PCR reaction system

Through optimizing each reaction conditions, finally confirmed SIV H9 hypotype, PCV2, the multiple SYBR Green of PRV I quantitative fluorescent PCR reaction system, in 25 μ L systems, add following composition:

The reaction parameter that this reaction is finally confirmed is: 95 ℃ of preparatory sex change 1min, 95 ℃ of 10s, 57 ℃ of 15s, 72 ℃ of 15s, 40 circulations.Reaction is heated to 95 ℃ earlier after finishing, and then reduces to 65 ℃, and beginning is incremented to 94 ℃ with 0.5 ℃/sec and detects fluorescent signal, and then draws the melting curve of amplified production.

2.5SIV confirming of the melting curve of H9 hypotype, PCV2, the triple SYBR Green of PRV I fluorescent quantitative PCR product and SIV H9 hypotype, PCV2, PRV TM value

According to SIV H9 hypotype, PCV2, the triple SYBR Green of PRV I quantitative fluorescent PCR reaction conditions; Recombinant plasmid with SIV H9 hypotype, PCV2, PRV is a template; Carry out the reaction of fluorescent PCR quantitative PCR;, software Agilent Mx3005P obtains melting curve (Fig. 4) after analyzing, as can be seen from the figure three TM values that the pairing temperature of specific peak value is SIV H9 hypotype, PCV2, PRV.Through after the compiling of a plurality of batches of testing datas, finally confirmed the TM value of SIV H9 hypotype, PCV2, three kinds of viruses of PRV, be respectively: 91.7-92.0 ℃ of SIV H9 hypotype, PCV284.8-85.1 ℃, PRV89.3-89.5 ℃; Negative control does not have peak value and produces.

2.6 MULTIPLE COMPOSITE SYBR Green I quantitative fluorescent PCR specificity assay

Can find out that from SIV H9 hypotype, PCV2, the multiple SYBR Green of PRV I quantitative fluorescent PCR specificity test-results (Fig. 5) PRRSV, PPV and negative control all do not have the generation of specificity peak value in the control group.Have only SIV H9 hypotype, PCV2, PRV composite fluorescence PCR in the test group to produce specific three peak values.SIV H9 hypotype TM value is 91.8 ℃, and the PCV2TM value is 84.8 ℃, and PRV TM value is 89.3 ℃.

2.7 multiple SYBR Green I quantitative fluorescent PCR replica test result

2.7.1 the SIV H9 hypotype of same concentration, PCV2, the triple SYBR Green of PRV I quantitative fluorescent PCR replica test result

In three replica tests, the TM value of these three kinds of viruses is all comparatively stable.PRV, PPV and negative control all do not have the generation of specificity peak value (like table 2, Fig. 6) in the control group.SIV H9 hypotype, PCV2, the reaction of the triple SYBR Green of PRV I quantitative fluorescent PCR with concentration have good stability.

The same concentration replica test of the multiple SYBR Green of table 2 I real-time fluorescence PCR is analyzed

2.7.2 the SIV H9 hypotype of different concns, PCV2, PRV composite S YBR Green I quantitative fluorescent PCR replica test result

In the different concns replica test, the TM value of these several kinds viruses is all comparatively stable.PPV, CSFV and negative control all do not have the specificity peak value to produce (like table 3, Fig. 7-9) in the control group.The SIV H9 hypotype of different concns, PCV2, the reaction of PRV composite S YBRGreen I quantitative fluorescent PCR have good stability equally.

The different concns replica test of the multiple SYBR Green of table 3 I real-time fluorescence PCR is analyzed

2.8SIV, PCV2, the sensitivity test of PRV composite S YBR Green I quantitative fluorescent PCR test

The concentration of pGEM-H9, pGEM-PCV2 and three kinds of plasmids of pGEM-PRV is respectively 52.7 μ g/ μ L, 19.9 μ g/mL, 32.4 μ g/ μ L; After getting isopyknic two kinds of plasmid mixings, carry out 10 times of gradient dilutions, carry out the reaction of composite S YBRGreen I quantitative fluorescent PCR as template with this.The susceptibility of SIV recombinant plasmid can reach 184 copy/μ L, and the susceptibility of PCV2 recombinant plasmid can reach 207 copy/μ L, and the susceptibility of PRV recombinant plasmid can reach 193 copy/μ L.

2.9 triple fluorescent quantitative PCR and conventional PCR detect relatively

Doubtful trouble SIV, PCV2, PRV piglet are organized 23 parts of pathological material of disease (being numbered 1-23); 2 parts of negative control (being numbered 24,25) are extracted DNA or cDNA, carry out PCR and real-time PCR respectively and detect; The result shows; In 23 parts of pathological material of diseases, conventional PCR detects the positive pathological material of disease of 8 parts of SIV H9 hypotypes, and recall rate is 34.8%; 10 parts of positive pathological material of diseases of PCV2, recall rate is 43.5%; 10 parts of positive pathological material of diseases of PRV, recall rate is 43.5%; Triple fluorescent quantitative PCR can detect the positive pathological material of disease that conventional PCR detects equally, and the two identical rate reaches 100%; Among the triple fluorescent quantitative PCR, SIV H9 hypotype detects 13 parts of positive pathological material of diseases, and recall rate is 56.5%, and is higher by 21.7% than the recall rate of conventional PCR; PCV2 detects 14 parts of positive pathological material of diseases, and recall rate is 60.9%, and is higher by 17.4% than the recall rate of conventional PCR; PRV detects 14 parts of positive pathological material of diseases, and recall rate is 60.9%, is higher than conventional PCR than the high 17.4%.real-time PCR of the recall rate detection sensitivity of conventional PCR, can detect the non-detectable pathological material of disease of conventional PCR, and all can not detect for negative sample equally.

Table 4 SIV, PCV2, PRV composite fluorescence quantifying PCR method and conventional PCR method are relatively

Claims (2)

1.PCV2, PRV and the multiple SYBR Green of SIV H9 hypotype I real-time fluorescence PCR primer, it is characterized in that:

The sequence of SIV H9 hypotype primer is following:

Upstream primer P1:5 '-GGAACAACGCTTACCCTG-3 ';

Downstream primer P2:5 '-GAGACCATTGACAAGAGGC-3 ';

The sequence of PRV primer is following:

Upstream primer P3:5 '-GGCATCGGCGACTACCT-3 ';

Downstream primer P4:5 '-CGTCGTGCAGCGTGTAGAG-3 ';

The sequence of PCV2 primer is following:

Upstream primer P5:5 '-GGGCCAGAATTCAACCTTACCC-3 ';

Downstream primer P6:5 '-CGCACCTTCGGATATACTGTCA-3 '.

2. utilize the said primer of claim 1 to detect the multiple SYBR Green I real-time fluorescence PCR detection method of PCV2, PRV and SIV H9 hypotype; After comprising the DNA that extracts virus; By following SYBR Green I real-time fluorescence PCR reaction system and reaction conditions virus is detected, it is characterized in that:

Said SYBR Green I real-time fluorescence PCR reaction system adds following composition in 25 μ l systems:

Said reaction conditions is: 95 ℃ of preparatory sex change 1min; Get into circulation, 95 ℃ of sex change 10s, 58 ℃ of annealing 15s, 72 ℃ are extended 15s; 40 circulations are warming up to 95 ℃ of 15s after the loop ends, reduce to 65 ℃ of 15s again; Begin to be incremented to 94 ℃, gather the solubility curve that fluorescent signal draws amplified production, finish reaction in 95 ℃ of 15s from 65 ℃.

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