CN101260442B - Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus - Google Patents
Multiple real time fluorescence quantifying PCR method for detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and classical swine fever virus Download PDFInfo
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Abstract
The invention discloses a multiplex real-time fluorescent quantitation PCR method capable of detecting porcine circovirus, porcine parvovirus, porcine pseudorabies virus and hog cholera virus at the same time. The method of the invention detects that the four virus are all in good linear relations at the same time, all ten times serial dilution points of constructed normal plasmid are all in one straight line, CT value and copy number are in good linear relation, and regression analysis shows that the related coefficient of the CT value and the copy number is R<2> more than 0.99.The multiplexreal-time fluorescent quantitation PCR method has the advantages of excellent specificity, sensibility and stability, which can rapidly, sensitively and differentially detect the four virus with serious harm to the economy and can be used for early diagnosis of virus infection.
Description
Technical field
The present invention relates to a kind of detection method of virus, relate in particular to a kind of multiple real time fluorescence quantifying PCR method that detects pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus), Pestivirus suis simultaneously, belong to biological technical field.
Background technology
PRV (Pseudorabies virus) (Porcine pseudorabies virus, PRV), pig parvoviral (Porcineparvovirus, PPV), II type pig circular ring virus (Porcine circoviruses, PCV2), Pestivirus suis (Hog choleravirus, HCV) etc. all belong to the sick and breeding dysfunction syndrome cause of disease of porcine respiratory, and polyinfection often appears in 4 kinds of viruses.PCV-2 also with porcine respiratory disease syndrome (Porcine respiratory disease complex, PRDC), ablactation piglet multisystemic exhaustion syndrome (Postweaning multisystemic wasting syndrome, PMWS) and pigskin is scorching and nephrotic syndrome (Porcine dermatitis and nephropathy syndrome, PDNS) etc. all has close dependency, be former (the Segales J of important protopathy that causes multiple syndromes, Allan GM, DomingoM.Porcine circovirus diseases.Anim Health Res Rev.2005Dec; 6 (2): 119-42; Lipej Z, Segales J, Toplak I, et al.Postweaning multisystemic wasting syndrome (PMWS) in pigsin Croatia:detection and characterisation of porcine circovirus type2 (PCV2) .Acta VetHung.2005; 53 (3): 385-96; Sipos W, Duvigneau JC, Pietschmann P, et al.Porcinedermatitis and nephropathy syndrome (PDNS) is associated with a systemic cytokineexpression profile indicative of proinflammation and a Thl bias.Vet ImmunolImmunopathol.2005Sep15; 107 (3-4): 303-13; Kim J, Chung HK, Chae C.Association ofporcine circovirus2 with porcine respiratory disease complex.Vet is J.2003Nov; 166 (3): 251-6.), PRV causes that the ill mortality ratio of sucking pig is up to 100%.The no medicine treatment of ruminating animal morbidity, all death comes to an end; Swine fever is dispersed throughout the whole world, has the height contagiousness, is one of 15 kinds of Notifiable diseases of category-A of the bright row of World Organization for Animal Health; PPV found when carrying out the Pestivirus suis tissue culture in 1966, was the important cause of disease of pig breeding dysfunction.
At present, the method that detects various viruses is a lot, as the method that detects the PRV latent infection has the activation of latent virus in serological test, the body and detection, tissue block to cultivate and be total to culture technique, nucleic acid hybridization and conventional PCR detection technique, wherein conventional PCR detection technique susceptibility, specificity, stability and operability are all relatively good, but there is the harm to the operator such as crossed contamination and EB in the reacted processing of conventional PCR.And the real-time fluorescence quantitative PCR detection technique has just been filled up above defective, no matter be under study for action or in the detection of clinical sample, quick, the responsive characteristic of real-time fluorescence quantitative PCR makes it become effective ways (the Kim J that is applied to detect microorganism, ChungHK, Chae C.Association of porcine circovirus2 with porcine respiratory diseasecomplex.Vet is J.2003Nov; 166 (3): 251-6.), real-time fluorescence quantitative PCR fluorescent antibody test FA, IPMA method and isolation of virus more in the past has quick, responsive, safe more characteristics.The sensitivity of its detection is better than traditional detection method, and (Chae C.Association of porcinecircovirus2 with porcine respiratory disease complex.Vet J.2003Nov for Kim J, Chung HK; 166 (3): 251-6.), relatively PCR method susceptibility reaches doubly (Gunson RN of 10-100, Collins TC, Carman WF.Practicalexperience of high throughput real time PCR in the routine diagnostic virology setting.JClin Virol.2006Apr; 35 (4): 355-67.Epub2006Feb7).
Set up quick simultaneously, the responsive and accurate quantitative methods of 4 kinds of viruses, various etiological diagnosis and various cause of disease Study of Etiology are had great importance.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, a kind of multiple real time fluorescence quantifying PCR method that can detect pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus), Pestivirus suis simultaneously is provided, this method can detect quick, responsive, specifically to four kinds of serious viruses of economy harm, have good specificity, susceptibility and stability, can use the diagnosis in early stage of four kinds of virus infectiones.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of multiple real time fluorescence quantifying PCR method that detects pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus), Pestivirus suis simultaneously, comprise the standard plasmid for preparing 4 kinds of viruses and set up typical curve, determine to judge the standard of sample positive or negative, design detects the Tm value that primer carries out the fluorescent quantitative PCR of sample and obtains sample, the Tm value that is obtained is passed judgment on the steps such as positive or negative of sample with determined criterion, wherein, the establishment method of described typical curve is as follows:
(1) extracts the nucleic acid of pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus) and Pestivirus suis respectively according to ordinary method;
(2) be primer with SEQ ID NO:1 and SEQ ID NO:2, with II type pig circular ring virus (PCV2) nucleic acid that extracted is template, carry out pcr amplification according to ordinary method, product recovery, purifying with amplification, be connected on the pMD18-T carrier, transform, extract plasmid DNA, get II type pig circular ring virus standard plasmid;
With SEQ ID NO:3 and SEQ ID NO:4 is primer, is template with pig parvoviral (PPV) nucleic acid that is extracted, and carries out pcr amplification according to ordinary method, product recovery, purifying with amplification are connected on the pMD18-T carrier, transform, extract plasmid DNA, get the pig parvoviral standard plasmid;
With SEQ ID NO:5 and SEQ ID NO:6 is primer, with PRV (Pseudorabies virus) (PRV) nucleic acid that extracted is template, carry out pcr amplification according to ordinary method, product recovery, purifying with amplification, be connected on the pMD18-T carrier, transform, extract plasmid DNA, get the PRV (Pseudorabies virus) standard plasmid;
With SEQ ID NO:7 and SEQ ID NO:8 is primer, is template with Pestivirus suis (HCV) nucleic acid that is extracted, and carries out pcr amplification according to ordinary method, product recovery, purifying with amplification are connected on the pMD18-T carrier, transform, extract plasmid DNA, get the Pestivirus suis standard plasmid;
(3) 4 kinds of step (2) is prepared standard plasmids are made 10 times of serial dilutions respectively, with the diluent is template, carries out real-time fluorescence quantitative PCR, gathers fluorescent signal, to generate and to count logarithm with starting template be the x axle to machine software Rotor-gene6.0 as calculated, and the CT value is the typical curve of y axle.
The standard of described definite judgement positive or negative is: the CT value 35 the circulation in; The Tm value is respectively: PCV-2:86.69 ± 0.21; PPV:79.97 ± 0.15; PRV:88.56 ± 0.15; HCV:84.61 ± 0.16.
Described detection primer is: the primer that detects II type pig circular ring virus (PCV2) is SEQ ID NO:9 and SEQID NO:10; The primer that detects pig parvoviral (PPV) is SEQ ID NO:11 and SEQ ID NO:12; The primer that detects PRV (Pseudorabies virus) (PRV) is SEQ ID NO:13 and SEQ ID NO:14; The primer that detects Pestivirus suis (HCV) is SEQ ID NO:15 and SEQ ID NO:16.
Preferably, the typical curve of described detection II type pig circular ring virus is shown in Figure 3; The typical curve that detects pig parvoviral virus is shown in Figure 4; The typical curve that detects PRV (Pseudorabies virus) is shown in Figure 5; The typical curve that detects Pestivirus suis is shown in Figure 6.
The inventive method (SyMuReTi-PCR) is can be high responsive, special, detect PRV, PPV, PCV-2, HC rapidly easily simultaneously.Making up primer and detecting on the design of primers, download nucleotide sequence at GENEBANK, choose the conservative region design construction primer of PRV, PPV, the representative gE of PCV-2, HC, VP2, ORF2, raq gene respectively and detect the primer primer, compare among the BLAST among the NCBI, find that PCV-2 separates malicious nucleic acid with 195 strain PCV-2 and has 100% homology, amplified production 135nt separates malicious nucleic acid 100% homology with 100 strains of PCV-2, do not see point mutation, and do not have any homology with PCV-1; HC detects primer and 100 strain strains, 100% homology; PPV detects primer and 67 strain strains have 100% homology; PRV is no any with former with other viruses.The conservative property that detects design of primers has determined sample detection specificity and stability qualitatively, supports the SyMuReTi-PCR method to have good linear relationship simultaneously; Then determined quantitative accuracy.
The inventive method (SyMuReTi-PCR) detects uses the SYBR GREEN fluorescence dye thing that serves as a mark in the method for PCV-2, because of its cost is lower, more be applicable to the detection of animal doctor's cause of disease; In the PCR reaction system, add an amount of SYBR fluorescence dye, after the SYBR fluorescence dye mixes the dna double chain specifically, the emitting fluorescence signal, and do not mix SYBR dye molecule fluorescent signal emitted in the chain detect less than, thereby the increase that guarantees the increase of fluorescent signal and PCR product is synchronous fully.Therefore it uses stability better, can't occur detecting limited along with the variation of nucleic acid sequence origination point.
The present invention detects 4 kinds of viral methods (SyMuReTi-PCR) simultaneously and has good specificity, susceptibility and stability.Detect 4 kinds of viral no cross reactions simultaneously, judge that whether significantly or not SyMuReTi-PCR detects 4 kinds of viral method specificity T m value Statistics Application softwares simultaneously and judge; Batch between and batch in replica test show CV all less than 2% all in stable range; 4 kinds of cell culture and virus susceptibilitys all are higher than 100 times of regular-PCRs, 4 kinds of lowest limit differences that different cell toxicants detect, and PCV-2 detects 0.00001TCID
50, PPV detects 0.001TCID
50, PRV detects 0.01TCID
50, HC detects 0.1TCID
50Characteristic, the cell toxicant of this and cell toxicant nucleic acid is pathogenic and cultural characters is closely related, the cell that do not make that has as PCV-2 causes a disease, the lowest limit height that detects, and 3 kinds of dna virus are to use the process that boiling method is not carried nucleic acid, do not consider the rate of loss problem, so the lowest limit of 3 kinds of dna virus detections is higher than HC.So SyMuReTi-PCR has the advantages such as defective that operation is more easy, sensitivity is higher and can effectively avoid regular-PCR to pollute.
The inventive method can be finished in 1.5hr.As seen, present method detection of being applied to the terrain clinical sample has good prospect.
The internal organs detection by quantitative that application the inventive method (SyMuReTi-PCR) is attacked malicious animal to experiment finds that the amount difference that different internal organs virus distributes is bigger.Bigger in inguinal lymphadenopathy poison abundance, be 10
5Copies/ul, it is less to distribute in duodenum, is 7 copy/ul.As seen can be applied to experimental study by SyReTi-PCR method of the present invention, assessment virus infection degree, monitoring virus infection level are further inquired into the pathogenic of virus, carry out etiologic etiological research.
In a word, the method that the SyMuReTi-PCR that the present invention set up detects various cause of diseases has good specificity, susceptibility and stability, can be applied to clinical etiological diagnosis and the Study of Etiology in the experimental study.
Description of drawings
Fig. 1 agarose gel electrophoresis detects pcr amplification product.
1, Marker DL2000; 2, the pcr amplification product 792bp of PCV; 3, the negative control of PCV; 4, MarkerDL2000; 5, the negative control of PRV; 6, the pcr amplification product 265bp of PRV; 7, Marker DL2000; 8, the pcr amplification product 764bp of PPV; 9, the negative control of PPV; 10, the negative control of HCV; 11, the pcr amplification product 303bp of HCV; 12, Marker DL2000.
Fig. 2 agarose gel electrophoresis is identified plasmid standard.
1, Marker DL2000; 2, the pcr amplification product 792bp of plasmid PCV; 3, the negative control of plasmid PCV; 4, Marker DL2000; 5, the negative control of plasmid PRV; 6, the pcr amplification product 265bp of plasmid PRV; 7, Marker DL2000; 8, the pcr amplification product 764bp of plasmid PPV; 9, the negative control of plasmid PPV; 10, the negative control of plasmid HC; 11, the pcr amplification product 303bp of plasmid HCV; 12, Marker DL2000.
The typical curve of Fig. 3 PCV-2.
The typical curve of Fig. 4 PPV.
The typical curve of Fig. 5 PRV.
The typical curve of Fig. 6 HCV.
Fig. 7 SyMuReTi-PCR detects the specificity test-results of 4 kinds of viruses.
Be negative differential curve of solubility curve, X-axis is represented solvent temperature, and Y-axis is represented the differentiate of fluorescent signal value and temperature; BinA is the Tm value of PPV; BinB is the Tm value of HC; BinC is the Tm value of PCV-2; BinD is the Tm value of PRV.
Further describe preparation method of the present invention and beneficial effect by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
Test materials
1.1 strain II type pig circular ring virus (Porcine circoviruses, PCV2), PRV (Pseudorabies virus) (Porcinepseudorabies virus, PRV), pig parvoviral (Porcine parvovirus, PPV), Pestivirus suis (Hogcholera virus, HCV) each 1 strain;
1.2 13 parts of internal organs collections of sample to be checked are from test pig (being provided by professor Liu Changming of Harbin Veterinary Medicine Inst., China Academy of Agriculture); 47 parts of pathological material of diseases respectively from Heilungkiang, the pig farm on ground such as Jilin, Henan, Hunan gathers;
1.3 reagent pMD18-T carrier,
Premix Ex Taq
TM(Perfect Real Time), EASYDilution (TOKARA); The little extraction reagent kit of plasmid (sky is the epoch); Plasmid Miniprep Kit (AXYGEN); In Vitro Transcription T7 Kit, DNase I (TOKARA); TR1zol
RLS Reagent, ThermoScript II (Invitrogen).
1.4 instrument Rotor-gene3000 quantitative PCR instrument; Eppendorf Biophoto meter.
The preparation of embodiment 1 standard plasmid
The segmental preparation of 1 purpose is extracted the nucleic acid of PRV, PPV, PCV-2, HCV according to ordinary method, nucleic acid is stored in-20 ℃ standby.The design primer is used OLIGO6.24, compares design primer (table 1) according to the nucleotide sequence among the GENEBANK.As template, carry out pcr amplification with the PRV, the PPV that extract, PCV-2, HCV viral nucleic acid, estimate that amplified production length is 265bp, 764bp, 792bp303bp with the primer of table 1.The amplified production that obtains is carried out agarose gel electrophoresis, the results are shown in Figure 1.
The primer of table 1 amplification target gene is to sequence
The preparation of 2 standard plasmids is reclaimed the purpose fragment according to Plasmid Miniprep Kit working method, to reclaim product is connected on the pMD18-T carrier, transform by the bacillus coli DH 5 alpha competence, the picking white colony, identify, positive bacteria propagation is extracted plasmid DNA, detects the standard plasmid of four kinds of viruses as real-time fluorescence quantitative PCR.
With the extension amplification outcome of 265bp, 764bp, 792bp303bp in T-Vector, the preparation standard product.Through conversion, positive bacteria screening, evaluation, show to have obtained to contain the segmental plasmid T-PRV of purpose, T-PPV, T-PCV-2, T-HCV, the results are shown in Figure 2.
1.1 it is 4.97 * 10 that Eppendorf Biophoto meter quantitative criterion plasmid is used in the dilution of four kinds of viral standard plasmids
12Copies/ μ l, 3.44 * 10
12Copies/ μ l, 8.18 * 10
12Copies/ μ l, 7.98 * 10
12Copies/ μ l, with EASY Dilution diluent with the standard substance plasmid according to 1.0 * 10
8～1.0 * 10
1Copies/ μ l10 doubly dilutes ,-20 ℃ of preservations.
1.2 detect the objective gene sequencing of design of primers,, use OLIGO6.24 design four pairs of special primers (table 2) according to the sequence of measuring with clone among the embodiment 1.
The primer of table 2 amplification target gene is to sequence
1.3SyMuReTi-PCR in Axygen Scientific Quality centrifuge tube (200 μ l), add successively: add respectively in sterilization deionized water 9.5 μ l, dimethyl sulfoxide (DMSO) 1 μ l, each 0.5 μ l of 4 pairs of primers of table 2, the different centrifuge tube different dilution different virus standard substance 1 μ l,
Premix Ex Taq
TM12.5 μ l, reaction system 25ul.The standard substance of every kind of virus are placed on a row by the dilution on earth order of high dilution, are convenient to analyze.Reaction parameter is: 95 ℃, and 10sec; 95 ℃, 5sec, 60 ℃, 20sec, 45 circulations.
1.4 the typical curve of setting up 4 kinds of viruses of SyMuReTi-PCR detection is done 10 times of serial dilutions, 1.0 * 10 with containing PRV, PPV, HC, the segmental cloned plasmids of PCV-2 purpose as standard substance respectively with PRV, PPV, HC, PCV-2 plasmid
8～1.0 * 10Copies/ μ l, 1.0 * 10
8～1.0 * 10
2Copies/ μ l, 1.0 * 10
8～1.0 * 10Copies/ μ l, 1.0 * 10
9～1.0 * 10Copies/ μ l carries out real-time fluorescence quantitative PCR as template.Rotor-gene3000 quantitative PCR instrument is gathered fluorescent signal, and will to generate automatically and count logarithm with starting template be the x axle to machine software Rotor-gene6.0 as calculated, and the CT value is the typical curve (Fig. 3-6) of y axle.When analyzing wherein a kind of typical curve, with Rotor-gene6.0 software the standard substance and the sample of other three kinds of viruses are all hidden, so just can be at the typical curve of four kinds of viruses of different interface analysis.9 of the PCV standard substance dilution point all point-blank show and work as gene copy number 10 by analysis
9In～10 scopes, all be the good linear relation between detection thresholding and the copy number, regression analysis shows that the two relation conefficient is R
20.999, amplification efficiency reaches 97%; 8 dilution point of PRV standard substance all point-blank, and when gene copy number 10
8～10
2In the scope, all be the good linear relation between detection critical cycle number (CT) and the copy number, regression analysis shows that the two relation conefficient is R
20.996, amplification efficiency reaches 93%; 8 dilution point of HCV standard substance all point-blank, the two relation conefficient is R
20.996; The PPV standard substance according to 6 extent of dilution all point-blank, the two relation conefficient is R
20.994, amplification efficiency reaches 99%.
Test example 3 SyMuReTi-PCR methods of the present invention detect 4 kinds of viral sensitivity tests
The PCV-2 of PK-15 cell cultures is from 10
1TCID
50～10
-6 TCID
5010 times of dilutions of series, PRV is from 10
1TCID
50～10
-6 TCID
5010 times of dilutions of series, 10 times of dilutions of series, PPV is from 2
1Blood clotting valency～2
-610 times of dilutions of blood clotting valency series and HC are from 10
1TCID
50～10
-6 TCID
5010 times of dilutions of series, three kinds of dna virus directly boiled 5 minutes, and RNA viruses is extracted nucleic acid, reverse transcription. and carry out quantitative PCR, regular-PCR detection sensitivity respectively, relatively susceptibility the results are shown in Table 3.
Test-results shows that the susceptibility of quantitative detecting method of the present invention is higher than the about 10-100 of regular-PCR doubly.
Table 3 SyMuReTi-PCR detects the susceptibility of 4 kinds of viruses
Annotate: PPV
*Be with equal dilution blood clotting valency; +: the positive;-: feminine gender.TCID
50Be half cell culture infective dose.
Note：+：Positive；-：Negative.TCID
50Tissue?culture?infectious?dose
Test example 4 SyMuReTi-PCR of the present invention detect the specificity test of 4 kinds of viruses
With the primer of PRV with, PPV, HCV, PCV-2, SIV, PRRSV and the known positive, negative sample DNA/cDNA reaction; The primer of PPV and PRV, HCV, PCV-2, SIV, PRRSV and the known positive, negative sample DNA/cDNA react; The primer of HC and PRV, PPV, PCV-2, SIV, PRRSV and the known positive, negative sample DNA/cDNA react; The primer of PCV-2 and PRV, PPV,, SIV, PRRSV and the known positive, negative sample DNA/cDNA reaction; Carry out the test of real-time fluorescence quantitative PCR specific detection jointly.The specificity test-results shows: each 2 parts of DNA/cDNA standard plasmid of the positive of 4 kinds of viruses all have specific amplification, and the peak value of solubility curve is single; And there are not the fluorescent signal of amplification, no CT value, the peak value of no solubility curve (Fig. 3) with 3 kinds of cell culture and virus, PRRSV, SIV except that self that 4 kinds of primers increase respectively.The specificity test-results shows that the SRT-PCR specificity is good.The specificity that 4 kinds of virus detects can judge whether it is specific amplification (table 5) to the goal gene Tm value of 4 kinds of viruses by same interface, is respectively the Tm of PPV, HC, PCV-2, PRV from the visible BinA of Fig. 3, BinB, BinC, BinD.The Tm scope of PPV, HC, PCV-2, PRV is the Tm value by 100 parts of positive, calculate mean number and standard deviation with statistical method again, gained scope PPV is that 79.82-80.12, HC are that 84.45-84.77, PCV-2 are that 86.48-86.90, PRV are 88.41-88.71 (table 4).
The Tm value of table 4 solubility curve
Annotate; M is a mean number; SD is a standard deviation; Tm is a solvent temperature.
Table 5 SyMuReTi-PCR detects the specificity of 4 kinds of viruses
Annotate: mark
*It is cell culture and virus; +: the positive;-: feminine gender; YL544124, L1001, Y1104, D45, D46, D45xiao are positive to be checked.
Note：
*is?virus?ofcell；+：Positive；-：Negative；YL544124，L1001，Y1104，D45，D46，D45xiao?is?sample.
Test example 5 SyMuReTi-PCR of the present invention detect 4 kinds of virus stability tests
With PRV, PPV, PCV-2, HC, get respectively 5 parts of positive and 1 part of negative sample criticize respectively in, batch between revision test.Revision test is that 5 duplicate samples are repeated 3 times in a real-time fluorescence in batch, the results are shown in Table 6; Revision test is that (4 days at interval) carry out the real-time fluorescence quantitative PCR test in the test of 3 different times between batch, the results are shown in Table 6.4 kinds of viruses batch between, batch in the average coefficient of variation CV value of revision test all less than 2%;
Table 6 SyMuReTi-PCR detect 4 kinds of viruses batch in replica test and batch between the variation coefficient of replica test
Annotate: CV is the variation coefficient; Note:CV is coefficient ofvariability
Test example 6 SyMuReTi-PCR of the present invention detect 4 kinds of viral sample tests
Test sample amounts to 60 parts, wherein terrain is gathered 27 parts of whole bloods, and 13 parts in malicious pig internal organs are attacked in test, and 20 pathological material of diseases are gathered from the pig farm on ground such as Henan, Hunan respectively, extract nucleic acid according to conventional methods, use SyMuReTi-PCR method and the regular-PCR test sample set up.Sample to be checked is a whole blood, and performance has collections of merely hitting of the doubtful pig of the relevant eqpidemic disease clinical symptom of PRV, PPV, PCV-2, HC certainly, clinical mainly show as dorsal body setae fluffy and disorderly, lose the appetite, some chatter and respiratory symptom occur, or dysgenesia etc. occurs.Cutaneous necrosis is hemorrhage, and the purple plague purpura of soybean grain size is arranged, the enlargement of lymph node, and pulmonary congestion, extravasated blood, meat become, and intestines drench swollen (clinical symptom of ground swinerys such as Henan, Hunan).
The regular-PCR method detects with SyMuReTi-PCR detects and the results are shown in Table 7 and table 8.As can be seen from Table 7: the PCV-2 regular-PCR detects 39 parts of positives, and SyMuReTi-PCR of the present invention detects 45 parts of positives; The PRV regular-PCR detects 14 parts of positives, and SyMuReTi-PCR detects 19 parts of positives; The HC regular-PCR detects 3 parts of positives, and SyMuReTi-PCR detects 4 parts of positives; PPV regular-PCR and SyMuReTi-PCR detect 3 parts; HC and PCV-2 hybrid infection viruses have 3 parts; HC and PRV hybrid infection viruses have 1 part; PCV-2 and PRV polyinfection have 4 parts.
Table 7 SyMuReTi-PCR test sample result
Annotate: criterion is defined as: in CT value in 35 circulate,
The Tm value is respectively:
PCV-2：86.69±0.21
PPV：79.97±0.15
PRV：88.56±0.15
HCV：84.61±0.16。
Table 8 SyMuReTi-PCR detects the sample result of 4 kinds of viruses
Annotate :+: the positive;-: negative Note:+:Positive;-: Negative
Sequence table
＜110〉Scientia Agricultura Sinica research institute Harbin veterinary institute
＜120〉multiple real time fluorescence quantifying PCR method of detection pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus), Pestivirus suis
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Claims (1)
1. a cover detects the primer of pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus), Pestivirus suis simultaneously, by SEQ ID NO:1-SEQ ID NO:16 totally 16 primers form, its using method comprises: the standard plasmid of 4 kinds of viruses of preparation, design detects primer, set up typical curve, determine to judge sample male standard, carry out the fluorescent quantitative PCR of sample and obtain the Tm value of sample, the Tm value that obtained is passed judgment on the positive of sample with determined criterion, and the establishment method of described typical curve is as follows:
(1) extracts the nucleic acid of pig circular ring virus, pig parvoviral, PRV (Pseudorabies virus) and Pestivirus suis respectively according to ordinary method;
(2) be primer with SEQ ID NO:1 and SEQ ID NO:2, with the II type pig circular ring virus nucleic acid that extracted is template, carry out pcr amplification according to ordinary method, product recovery, purifying with amplification, be connected on the pMD18-T carrier, transform, extract plasmid DNA, get II type pig circular ring virus standard plasmid;
With SEQ ID NO:3 and SEQ ID NO:4 is primer, is template with the pig parvoviral nucleic acid that is extracted, and carries out pcr amplification according to ordinary method, product recovery, purifying with amplification are connected on the pMD18-T carrier, transform, extract plasmid DNA, get the pig parvoviral standard plasmid;
With SEQ ID NO:5 and SEQ ID NO:6 is primer, is template with the PRV (Pseudorabies virus) nucleic acid that is extracted, and carries out pcr amplification according to ordinary method, product recovery, purifying with amplification are connected on the pMD18-T carrier, transform, extract plasmid DNA, get the PRV (Pseudorabies virus) standard plasmid;
With SEQ ID NO:7 and SEQ ID NO:8 is primer, is template with the Pestivirus suis nucleic acid that is extracted, and carries out pcr amplification according to ordinary method, product recovery, purifying with amplification are connected on the pMD18-T carrier, transform, extract plasmid DNA, get the Pestivirus suis standard plasmid;
(3) 4 kinds of step (2) is prepared standard plasmids are made 10 times of serial dilutions respectively, with the diluent is template, with SEQ ID NO:9 and SEQ ID NO:10 is the primer that detects II type pig circular ring virus, with SEQ ID NO:11 and SEQ ID NO:12 is the primer that detects pig parvoviral, with SEQ ID NO:13 and SEQ ID NO:14 is the primer that detects PRV (Pseudorabies virus), with SEQ ID NO:15 and SEQ ID NO:16 is the primer that detects Pestivirus suis, carry out real-time fluorescence quantitative PCR, gather fluorescent signal, to generate and to count logarithm with starting template be the x axle to machine software Rotor-gene6.0 as calculated, and the CT value is the typical curve of y axle;
Described definite judgement male standard is: the CT value 35 the circulation in; The Tm value is respectively: II type pig circular ring virus: 86.69 ± 0.21; Pig parvoviral: 79.97 ± 0.15; PRV (Pseudorabies virus): 88.56 ± 0.15; Pestivirus suis: 84.61 ± 0.16.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6642033B1 (en) * | 1999-07-20 | 2003-11-04 | V.I. Technologies, Inc. | Nucleic acids for detecting parvovirus and methods of using same |
| CN1458167A (en) * | 2003-06-02 | 2003-11-26 | 中国农业科学院哈尔滨兽医研究所 | Truncated expressed porcine circovirus II capsid protein antigen and use thereof |
| CN1477108A (en) * | 2003-06-02 | 2004-02-25 | 中国农业科学院哈尔滨兽医研究所 | Recombinant gE protein and its application for diagnosis of pseudo-rabies virus infection |
-
2007
- 2007-03-09 CN CN2007100797663A patent/CN101260442B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6642033B1 (en) * | 1999-07-20 | 2003-11-04 | V.I. Technologies, Inc. | Nucleic acids for detecting parvovirus and methods of using same |
| CN1458167A (en) * | 2003-06-02 | 2003-11-26 | 中国农业科学院哈尔滨兽医研究所 | Truncated expressed porcine circovirus II capsid protein antigen and use thereof |
| CN1477108A (en) * | 2003-06-02 | 2004-02-25 | 中国农业科学院哈尔滨兽医研究所 | Recombinant gE protein and its application for diagnosis of pseudo-rabies virus infection |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857904B (en) * | 2009-04-10 | 2013-10-16 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Multiple real-time fluorescence PCR (Polymerase Chain Reaction) detection method and kit of soybean quarantine virus diseases |
| CN104561368A (en) * | 2014-09-30 | 2015-04-29 | 浙江理工大学 | Multiple real-time fluorescent quantative PCR rapid diagnosis kit for six porcine viruses |
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