CN102071257B - Dual SYBR Green I real-time fluorescence PCR detection primer and method for porcine parvovirus and porcine circovirus type 2 - Google Patents
Dual SYBR Green I real-time fluorescence PCR detection primer and method for porcine parvovirus and porcine circovirus type 2 Download PDFInfo
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Abstract
The invention discloses a dual SYBR Green I real-time fluorescence polymerase chain reaction (PCR) detection primer and a dual SYBR Green I real-time fluorescence PCR detection method for porcine parvovirus and porcine circovirus type 2. The primer is designed and synthesized, and the dual SYBR Green I real-time fluorescence PCR detection method for the porcine parvovirus and the porcine circovirus type 2 by using the primer comprises the following steps of: extracting deoxyribose nucleic acid (DNA) of a sample; and detecting the sample according to an SYBR Green I real-time fluorescence PCR reaction system and an SYBR Green I real-time fluorescence PCR amplification program. By the primer and the method, two viruses, namely the porcine parvovirus and the porcine circovirus type 2 can be detected simultaneously, and porcine pseudorabies virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus cannot be detected. The primer and the method have the characteristics of higher specificity, repeatability and sensitivity.
Description
Technical field
The present invention relates to a kind of real-time fluorescence PCR detection method, be specifically related to pig parvoviral and porcine circovirus 2 type two-fold SYBR Green I real-time fluorescence PCR detection method.
Background technology
Porcine parvovirus (PPV) is that to infect what cause by pig parvoviral be the viral infectious of principal character with the sow breeding difficulty; It is characterized in that sow pregnant in earlier stage infects should virus the time; Through placenta embryo or fetus are attacked; Cause sow miscarriage, embryonic death, fetal anomaly, fetus mummification and infertile etc., also can cause dermatitis and the diarrhoea of piglet simultaneously, but the pig of other type infects the no obvious clinical symptom in back.PPV recall rate in swinery is very high, and the positive rate of antibody in swinery reaches 50%～80%, is one of main diseases toxicity breeding difficulty property disease of puzzlement pig industry development.
At present; Above virus a large amount of correlative studys have been carried out both at home and abroad; Set up methods such as viral separation, immunofluorescence technique, ELISA method, immunohistochemical methods method, and the multiple PCR method that detects PPV, PCV2, like competitive PCR, sleeve type PCR and in situ hybridization etc.But it is consuming time that virus is separated, though in situ hybridization has very high sensitivity, and complex operation, cost is higher, and the cycle is longer, and having relatively high expectations to pathological material of disease.PCR has higher susceptibility and specificity at present, is widely used in clinical diagnosis.But because difference is bigger between the different primers, and the influence of various factors during pcr amplification, false-negative result often appears during diagnosis easily, and can not be quantitative; Especially to the often omission of inapparent infection animal, thereby caused the popular of disease and spread.Though the TaqMan probe method that PRV, PPV etc. set up can be qualitative, quantitative, cost is higher, is unfavorable for applying.SYBR Green I is the non-specific optical dye that is incorporated into double-stranded DNA, can combine with multiple extension increasing sequence.SYBR Green I PCR detection method can design and synthesize the technology of the special any gene of the direct augmentation detection of fluorescently-labeled probe.
Summary of the invention
The technical problem that the present invention will solve be the design with synthesize pig parvoviral and porcine circovirus 2 type primer after, utilize primer to detect pig parvoviral and two kinds of viruses of porcine circovirus 2 type simultaneously with SYBR Green I real-time fluorescence PCR detection method.
Technical scheme of the present invention is: the sequence of pig parvoviral and porcine circovirus 2 type two-fold SYBR Green I real-time fluorescent PCR testing primer is following:
The sequence of porcine circovirus 2 type primer is following:
Upstream primer P1:5 '-TAA CTA CTC CTC CCG CCA TAC-3 ';
Downstream primer P2:5 '-GCC TAC GTG GTC TAC ATT TCC-3 ';
The sequence of pig parvoviral primer is following:
Upstream primer P3:5 '-TGG GAG GGC TTG GTT AGA-3 ';
Downstream primer P4:5 '-TGG TGG TGA GGT TGC TGA T-3 '.
Utilize above-mentioned primer to detect the double SYBR Green I real-time fluorescence PCR detection method of pig parvoviral and porcine circovirus 2 type; After comprising the DNA that extracts sample, sample is detected by following SYBR Green I real-time fluorescence PCR reaction system and SYBRGreen I real-time fluorescence PCR amplification program.
Said SYBR Green I real-time fluorescence PCR reaction system adds following composition in 25 μ l systems:
SYBR?Green?I?PreMix 15μl
P1/P3 0.5+0.5μl
P2/P4 0.5+0.5μl
ddH
2O 6μl
25μl
Said SYBR Green I real-time fluorescence PCR amplification program is: 95 ℃ of preparatory sex change 5min; Get into circulation, 95 ℃ of sex change 15s, 55 ℃ of annealing 20s, 72 ℃ are extended 20s, 40 circulations; Reaction is heated to 95 ℃ earlier after finishing, and then reduces to 65 ℃, and beginning is incremented to 95 ℃ with 0.5 ℃/s and detects the solubility curve that fluorescent signal draws amplified production.
The invention has the beneficial effects as follows: the present invention can detect pig parvoviral and two kinds of dna virus of porcine circovirus 2 type simultaneously, can copy the porcine circovirus 2 type plasmids by minimum detection 175 copy pig parvoviral plasmids and 156; Can not detect PRV (PRV), CSFV, porcine reproductive and respiratory syndrome virus and swine influenza virus.The present invention has specificity, repeatability and susceptibility preferably, for the detection of PPV, PCV2 provides a kind of new method, can be used as the purification detection method of large-scale pig farm PPV, PCV2; Set up the swinery of no PPV, PCV2; Simultaneously also be the Molecule Epidemiology Investigation that PPV, PCV2 infect, research PPV, PCV2 molecular pathogenesis, early diagnosis; The development of diagnostic kit and exploitation, the immunologic mechanism of medicine or vaccine provides test basis and technique means.
Description of drawings
Fig. 1 is a PCV2ORF2 portion gene plasmid PCR qualification result;
Fig. 2 is a PPVVP2 portion gene plasmid PCR qualification result;
Wherein, Fig. 1, among Fig. 2,1. purpose fragment, M.DL 2000Marker;
Fig. 3 is double SYBR Green I real-time fluorescence PCR reaction solubility curve;
Fig. 4 is double SYBR Green I real-time fluorescence PCR atopic test solubility curve;
Fig. 5 is that double SYBR Green I real-time fluorescence PCR reaction is with concentration replica test solubility curve;
Fig. 6 is double SYBR Green I real-time fluorescence PCR reaction different concns replica test solubility curve.
Embodiment
Embodiment
The sequence of pig parvoviral and porcine circovirus 2 type two-fold SYBR Green I real-time fluorescent PCR testing primer is following:
The sequence of porcine circovirus 2 type primer is following:
Upstream primer P1:5 '-TAA CTA CTC CTC CCG CCA TAC-3 ';
Downstream primer P2:5 '-GCC TAC GTG GTC TAC ATT TCC-3 ';
The sequence of pig parvoviral primer is following:
Upstream primer P3:5 '-TGG GAG GGC TTG GTT AGA-3 ';
Downstream primer P4:5 '-TGG TGG TGA GGT TGC TGA T-3 '.
Utilize above-mentioned primer to detect the double SYBR Green I real-time fluorescence PCR detection method of pig parvoviral and porcine circovirus 2 type; After comprising the DNA that extracts sample, sample is detected by following SYBR Green I real-time fluorescence PCR reaction system and SYBRGreen I real-time fluorescence PCR amplification program.
Said SYBR Green I real-time fluorescence PCR reaction system adds following composition in 25 μ l systems:
SYBR?Green?I?PreMix 15μl
P1/P3 0.5+0.5μl
P2/P4 0.5+0.5μl
ddH
2O 6μl
25μl
Said SYBR Green I real-time fluorescence PCR amplification program is: 95 ℃ of preparatory sex change 5min; Get into circulation, 95 ℃ of sex change 15s, 55 ℃ of annealing 20s, 72 ℃ are extended 20s, 40 circulations; Reaction is heated to 95 ℃ earlier after finishing, and then reduces to 65 ℃, and beginning is incremented to 95 ℃ with 0.5 ℃/s and detects the solubility curve that fluorescent signal draws amplified production.
In the foregoing description, about materials and methods
1 materials and methods
1.1 material
1.1.1 strain, cell strain and bacterial strain
The PK-15 cell, PRV (PRV) standard strain is all available from China Veterinary Drugs Supervisory Inst.; Porcine circovirus 2 type, the pig parvoviral standard strain, porcine reproductive and respiratory syndrome virus, CSFV, the swine influenza virus standard strain is all available from Henan Province's animal food safety key lab; Engineering bacteria DH5 α competent cell is available from the precious biotechnology in Dalian ltd.
1.1.2 solution commonly used and substratum
The LB liquid nutrient medium; Penbritin (Ampicillin, Amp) stock solution, 100mg/ml; IPTG liquid concentration is 100mg/ml; X-gal concentration is 20mg/ml, is stored in the brown bottle or in the bottle with the aluminium foil parcel, above solution all be stored in-20 ℃ subsequent use.
1.1.3 key instrument and reagent
The ultraviolet gel imaging system is available from U.S. ALPHAINNOTECH company; Rotor-Gene 3000 type real-time quantitative PCRs amplifications appearance available from Australian genome company, PTC-200 type PCR appearance available from U.S. MJ company, 3K30 type high speed freezing centrifuge available from German SIGMA company etc.
SYBR Green I PreMix; EX TaqDNA polysaccharase, DNA marker DL2000 are available from the precious biotechnology in Dalian ltd; Protein K is available from Huamei Bio-Engrg Co.; The DMEM substratum is available from GIBCOBRL company; Foetal calf serum is available from Hyclone company; Trypsinase is available from LTI company; T4DNA ligase enzyme, PGEM-T Easy plasmid vector are all available from Promega company; Dna gel reclaims test kit available from the clean Bioisystech Co., Ltd of Hangzhou Wei Te; Plasmid extraction kit is available from vast Tyke, Beijing biotech firm.
1.2 method
1.2.1PCV2 multiplication by culture
PCV2 is inoculated in not adherent PK-15 cell synchronously, connects the poison amount, cell is placed 37 ℃, 5%CO for 1/10 of nutrient solution
2Incubator in cultivate 72h after, stop cultivating and also receive poison.Multigelation 2～3 times is collected viral liquid, and extraction or-80 ℃ of storages of directly carrying out viral RNA are subsequent use.
1.2.2PPV multiplication by culture
The PK-15 cell that will be in logarithmic phase with 0.25% trysinization after, put into 37 ℃, 5%CO
2Incubator in cultivate.Treat that cell attachment grows to 60% of growth surface～70% o'clock, after taking out culturing bottle and washing 2～3 times with PBS liquid, it is viral that the inoculum size of press nutrient solution 1/10 volume is inoculated PPV, then cell placed 37 ℃, 5%CO
2Incubator in cultivate.Every day is the observation of cell pathology under inverted microscope, receives poison behind the cultivation 72h.With the viral liquid multigelation of collecting 3 times, extraction or-70 ℃ of storages of directly carrying out viral DNA are subsequent use.
1.2.3 design of primers is with synthetic
According to pig parvoviral NADL-2 strain (NC001718) the VP2 gene order of GenBank login, according to the design of primers principle, Using P rimer Premier 5.0 biosoftwares are designed 1 pair of special primer, and the expectation amplified fragments is 313bp.Primer sequence is following:
Upstream primer P1:5 '-TGG GAG GGC TTG GTT AGA-3 ';
Downstream primer P2:5 '-TGG TGG TGA GGT TGC TGA T-3 ';
The porcine circovirus 2 type ORF2 gene of announcing with GenBank is canonical sequence (AF027217), designs 1 pair of upstream primer and downstream primer with Primer Premier5.0 biosoftware, and the expectation amplified fragments is 171bp.The sequence of primer is following:
Upstream primer P3:5 '-TAA CTA CTC CTC CCG CCA TAC-3 ';
Downstream primer P4:5 '-GCC TAC GTG GTC TAC ATT TCC-3 ';
Above-mentioned primer is given birth to worker's biotechnology ltd by Shanghai and is synthesized.
1.2.4PCV2, the extraction of PPV, PRV viral DNA
Traditional Proteinase K process for extracting: get each 450 μ l of viral liquid of propagation, 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) again; 56 ℃ of water-bath 1h; Add isopyknic balance phenol then, mixing, the centrifugal 5min of 10000rpm.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 10min of 12000rpm, 70% washing with alcohol 2 times, drying adds the dissolving of 25 μ l ultrapure waters, be stored in-20 ℃ subsequent use.
1.2.5 the preparation of porcine reproductive and respiratory syndrome virus, CSFV and swine influenza virus cDNA
Get the viral liquid of 400 μ l respectively, add 400 μ l Trizol, thermal agitation; Add 100 μ l chloroforms: primary isoamyl alcohol (24: 1) thermal agitation 30s, mixing; Put into desk centrifuge 12000rpm, the centrifugal 5min of room temperature; Get the upper strata water, carefully be transferred in the aseptic centrifuge tube of the 1.5ml of DEPC water treatment, add 150 μ l absolute ethyl alcohols, mixing; Solution in the last step is all transferred in the UnLQ-10 pillar, and room temperature is placed 2min, makes the RNA in the solution combine 8000rpm, centrifugal 1min as much as possible with the filter membrane of pillar; Take out pillar and discard the waste liquid in the collection tube, pillar is put into collection tube and added 450 μ lRPE Solution, room temperature leaves standstill 2min, 10000rpm, and centrifugal 30s, and repeat once; Take out pillar, discard waste liquid, 12000rpm, empty from 3min; Take out pillar, put into the aseptic centrifuge tube through the 1.5ml of DEPC water treatment, central authorities add 16 μ l DEPC water in the post inner membrance, place 2min for 55～80 ℃; 10000rpm, centrifugal 1min, solution in the collection tube wherein contains the RNA of extractive to some extent three kinds of viruses, can use immediately or-70 ℃ of storages subsequent use.
In the RNA of three kinds of viruses extracting, add reverse transcription random primer (10pmol/ μ l) respectively, 4 μ l dNTP (2.5mmol/L) place 5min, ice bath 2min for 70 ℃; Add 4 μ l reverse transcription Buffer, 2 μ l 0.1M DTT, 1 μ l Rnaseinfnibitor (40U/ μ l) places 2min for 42 ℃; Add 1 μ lM-MLV ThermoScript II (200U/ μ l), 42 ℃ of effect 1h place 15min, deactivation ThermoScript II for 70 ℃.CDNA after the reverse transcription is subsequent use in-20 ℃ of storages.
1.2.6 the preparation of plasmid template standard substance
PCV2DNA and PPV DNA to extract are template, with optimized conditions amplification PCV2 ORF2 gene fragment and PPV VP2 gene fragment.In 50 μ l reaction systems, add successively: 10 * PCR damping fluid, 5 μ l, MgCl
2(3mmol/L) 1.5 μ l, dNTPs concentration is (10mmol/L) 1.8 μ l, Taq enzyme 1U; Each 0.5 μ l of upstream and downstream primer (50pmol/L), template 5 μ l manage EP to 50 μ l. with the distilled water benefit at last and put the PCR appearance; Increase by following program: behind 95 ℃ of preparatory sex change 5min, advance the people 95 ℃ of 30s that circulate, 55 ℃ of 30s; 72 ℃ of 40s, after 30 circulations, 72 ℃ are extended 10min; 4 ℃ are finished reaction.2.5% agarose gel electrophoresis.
To amplify the purpose fragment and reclaim the test kit recovery by glue; Connecting test kit according to carrier is connected the purpose fragment with the pGEM-TEasy carrier; Transform DH5 α competent cell, select positive bacterium colony, carry out after bacterium liquid PCR identifies; Extract DNA with plasmid extraction kit, send Dalian precious biotechnology ltd to check order.Through the positive plasmid of sequence verification as the template standard article, and called after pGEM-VP2 and pGEM-PCV2 respectively.
1.2.7 the optimization of double SYBR Green I real-time fluorescence PCR reaction system condition
SYBR Green I real-time fluorescence PCR reaction system is following:
In 25 μ l systems, add following composition:
SYBR?Green?I?PreMix 13μl
P1/P3 0.5+0.5μl
P2/P4 0.5+0.5μl
ddH
2O 8μl
25μl
Instantaneous centrifugal after, EP pipe is placed 95 ℃ of preparatory sex change 5min on the quantitative real time PCR Instrument; Get into circulation, 95 ℃ of sex change 15s, 55 ℃ of annealing 20s, 72 ℃ are extended 20s, 40 circulations; Reaction is heated to 95 ℃ earlier after finishing, and then reduces to 65 ℃, and beginning is incremented to 95 ℃ with 0.5 ℃/s and detects the solubility curve that fluorescent signal draws amplified production.Set up the negative control of no template simultaneously.The optimization of this test reaction conditions is under the condition of two kinds of virus particle template equivalent, to carry out.
1.2.7.1 the optimization of primer concentration
Primer concentration 0.2 μ l, 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 reaction of SYBR Green I real-time fluorescence PCR respectively, to choose the best primer concentration of this virus of amplification.Get the primer amount of equal volume during interpolation.
1.2.7.2 the optimization of annealing temperature
Two-fold SYBR Green I real-time fluorescence PCR reacts with 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃ annealing temperature respectively, to confirm best annealing temperature.
1.2.7.3SYBR the optimization of PreMix concentration
The concentration of optical dye premix enzyme SYBR PreMix is 5U/ μ l in this test; The fixing TV of SYBR Green I real-time fluorescence PCR reaction system, the optimum concn of screening SYBR PreMix in the SYBRGreen I real-time fluorescence PCR reaction system through the addition that changes optical dye premix enzyme SYBR PreMix.Screen with the concentration of 10 μ l, 12.5 μ l, 15 μ l, 17.5 μ l, 20 μ l, 22.5 μ l respectively.
1.2.8 specificity check
According to SYBR Green I real-time fluorescence reaction system; Add PCV2, PPV cell toxicant DNA 1 μ l (about 20ng) respectively, porcine reproductive and respiratory syndrome virus (PRRSV) cell toxicant cDNA 1 μ l (about 20ng), swine influenza virus (SIV) cell toxicant cDNA 1 μ l (about 20ng); PRV (PRV) cell toxicant DNA 1 μ l (about 20ng); CSFV (CSFV) cell toxicant cDNA 1 μ l (about 20ng) establishes negative control simultaneously, verifies its specificity.
1.2.9 repeatability check
The plasmid standard of choosing PCV2, the same concentration of PPV carries out double SYBR Green I real-time fluorescence PCR reaction replica test, reaction repetition 3 times; The plasmid standard of different concns is carried out double SYBR Green I real-time fluorescence PCR reaction replica test, reaction repetition 3 times; Through the TM value of every kind of virus and the analysis of solubility curve, the stability of checking PPV, PCV2 two-fold SYBRGreen I real time fluorescent PCR method.
1.2.10 real-time fluorescence PCR sensitivity testing
Respectively the recombinant plasmid of PCV2, PPV is surveyed OD
260Be worth, calculate the copy number of every microlitre, carry out 10 times of gradient dilutions then, carry out double SYBR Green I real-time fluorescence PCR reaction as template, and then confirm the susceptibility of every kind of virus of double SYBR Green I real-time fluorescence PCR reaction detection with this.
1.2.11 doubtful PCV2, PPV samples detect the comparison that reaches with conventional PCR detection method
Get the pathological material of disease that the doubtful PCV2 that picks up from a plurality of districts and cities pig farm in the Henan Province, PPV infect and carry out double SYBRGreen I quantitative fluorescent PCR and detection, to confirm the practicality of this method.
2 results
2.1 plasmid pcr amplification
Like Fig. 1, PCV2ORF2 portion gene shown in Figure 2, PPV VP2 portion gene plasmid PCR qualification result have amplified respectively and the fragment of estimating big or small corresponding to 171bp and 313bp.PGEM-PCV2 and pGEM-VP2 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 respectively 99.6%, more than 99.7%.
The PPV, the PCV2 plasmid DNA concentration that record extraction are respectively: pGEM-VP2 mass concentration=141 μ g/ml, pGEM-PCV2 mass concentration=77.5 μ g/ml.Through calculating the pGEM-VP2 plasmid concentration is 7.86 * 10
13Copy/ml, pGEM-PCV plasmid concentration are 4.53 * 10
13Copy/ml.
2.2 the optimum result of double SYBR Green I real-time fluorescence PCR reaction conditions
Though SYBR Green I is a kind of no specific optical dye, when carrying out double PCR test, it is not that purpose fragments homogeneous and all combine, but can preferentially combine with some fragment.This just need carry out condition optimizing to reaction system, has the ratio of enough dyestuffs and various primers moderate in the guarantee system.
2.2.1 primer concentration optimum result
PCV2, PPV primer all adopt the amount of 50pmoL/ μ l 0.5 μ l in 25 μ l systems, to react, and can produce specific single peak value, and negative control does not have the specificity peak value and produces.The best primer concentration of PCV2, the reaction of PPV two-fold SYBR Green I real-time fluorescence PCR is 0.5 μ l.
2.2.2 annealing temperature optimum result
When 55 ℃ of PCV2, PPV annealing temperature, produced specific peak value.PPV Tm value is respectively 77.2 ℃, and 77.2 ℃, 77.2 ℃; The PCV2Tm value is respectively 82.2 ℃, and 81.7 ℃, 82 ℃. other temperature and negative control all do not have the specificity peak value and produce.The optimum annealing temperature of PPV, the reaction of PCV2 two-fold SYBR Green I real-time fluorescence PCR is 55 ℃.
2.2.2SYBR the optimum result of PreMix concentration
SYBR PreMix addition is when 15 μ l in the reaction of two-fold SYBR Green I real-time fluorescence PCR, and the two all can produce specific peak value, and negative control does not have the specificity peak value and produces.
2.3 confirming of double SYBR Green I real-time fluorescence PCR reaction system
Through the optimization of each reaction conditions, finally confirmed PCV2, PPV two-fold SYBR Green I real-time fluorescence PCR reaction system, in 25 μ l systems, add following composition:
SYBR?Green?I?PreMix 15μl
P1/P3 0.5+0.5μl
P2/P4 0.5+0.5μl
ddH
2O 6μl
25μl
The loop parameter of this reaction is finally confirmed as: 95 ℃ of preparatory sex change 5min; Get into circulation, 95 ℃ of sex change 15s, 55 ℃ of annealing 20s, 72 ℃ are extended 20s, 40 circulations; Reaction is heated to 95 ℃ earlier after finishing, and then reduces to 65 ℃, and beginning is incremented to 95 ℃ with 0.5 ℃/s and detects the solubility curve that fluorescent signal draws amplified production.
2.4 confirming of the solubility curve of double SYBR Green I real-time fluorescence PCR amplified production and PCV2, PPV Tm value
The present invention utilizes the difference of Tm value to carry out the differentiation of nucleic acid fragment, and the Tm value of nucleic acid fragment is main relevant with GC content, sequence length and sequential structure.The amplified fragments gene order GC content of PPV is lower than PCV2 amplified fragments gene order GC content among the present invention, and the Tm value of PCV2 amplified fragments is higher than the Tm value of the amplified fragments of PPV.According to PCV2, PPV two-fold SYBRGreen I real-time fluorescence PCR reaction conditions; Recombinant plasmid with PCV2, PPV is a template; Carry out the PCR reaction; After software Rotor-gen6.0 analyzes, obtain solubility curve, as shown in Figure 3, as can be seen from the figure two pairing temperature of specific peak value are the Tm value of PCV2, PPV.The Tm value of PCV2 amplified fragments and the Tm value of PPV amplified fragments are not changeless, and their Tm value can change in certain TR, and the Tm value variation range of PCV2 is 81.5～82.5 ℃, and the Tm value variation range of PPV is 77.2～77.5 ℃.The two differs 4～6 ℃, therefore, can utilize the difference of the two amplified production fragment Tm value and two specific peaks of solubility curve to diagnose and differentiate differentiation.
2.5 double SYBR Green I real-time fluorescence PCR specificity assay
As shown in Figure 4 from PCV2, PPV two-fold SYBR Green I real-time fluorescence PCR specificity test-results, as can be seen from the figure, PRRSV, PRV, SIV, CSFV and negative control all do not have the specificity peak value and produce in the control group.Have only PCV2, PPV two-fold real-time fluorescence PCR in the test group to produce specific two peak values.PCV2 Tm value is 81.7 ℃, and PPV Tm value is 77.4 ℃.
2.6 double SYBR Green I real-time fluorescence PCR replica test result
2.6.1 the PCV2 of same concentration, PPV two-fold SYBR Green I real-time fluorescence PCR replica test result
In 3 replica tests, each viral Tm value is all comparatively stable.Porcine reproductive and respiratory syndrome virus, CSFV and swine influenza virus, PRV and negative control all do not have the generation of specificity peak value in the control group.Like table 1, shown in Figure 5.PCV2, the reaction of PPV two-fold SYBR Green I real-time fluorescence PCR with concentration have good stability.
The same concentration replica test of the multiple SYBR Green of table 1 I real-time fluorescence PCR is analyzed
2.6.2 the PCV2 of different concns, PPV two-fold SYBR Green I real-time fluorescence PCR replica test result
In the different concns replica test, each viral TM value is all comparatively stable.Porcine reproductive and respiratory syndrome virus, CSFV and swine influenza virus, PRV and negative control all do not have the generation of specificity peak value in the control group, and be like table 2, shown in Figure 6.The PCV2 of different concns, the reaction of PPV two-fold SYBR Green I real-time fluorescence PCR have good stability equally.
The different concns replica test of the multiple SYBR Green2I of table 2 real-time fluorescence PCR is analyzed
2.7PCV2, the sensitivity test of PPV two-fold SYBR Green I real-time fluorescence PCR tests
The concentration of pGEM-A and two kinds of plasmids of pGEM-B is respectively 141 μ g/ml, 77.5 μ g/ml; After getting isopyknic two kinds of plasmid mixings, carry out 10 times of gradient dilutions, carry out double SYBR Green I real-time fluorescence PCR reaction as template with this.The susceptibility of PCV2 recombinant plasmid can reach 156 copy/μ l, and the susceptibility of PPV recombinant plasmid can reach 175 copy/μ l.
2.8 real-time fluorescence PCR and conventional PCR detect relatively
The piglet of doubtful trouble PCV2, PPV is organized 30 parts of pathological material of disease, after 2 parts of negative control are extracted DNA, carry out real-time fluorescence PCR and conventional PCR respectively and detect, in 30 parts of pathological material of diseases, conventional PCR detects the positive pathological material of disease of 14 parts of PPV, and recall rate is 46.7%; Detect the positive pathological material of disease of 13 parts of PCV2, recall rate is 43.3%; The two-fold real-time fluorescence PCR can detect the positive pathological material of disease that conventional PCR detects equally, and the two coincidence rate reaches 100%; In the two-fold real-time fluorescence PCR, PPV detects 24 parts of positive pathological material of diseases, and recall rate is 80%, and is higher by 33.3% than the recall rate of conventional PCR; PCV2 detects 27 parts of positive pathological material of diseases, and recall rate is 90%, and is higher by 46.7% than the recall rate of conventional PCR.The real-time fluorescence PCR detection sensitivity is higher than conventional PCR, can detect the non-detectable pathological material of disease of conventional PCR, and all can not detect for negative sample equally.
Claims (1)
1. pig parvoviral and porcine circovirus 2 type two-fold SYBR Green I real-time fluorescent PCR testing primer is characterized in that:
The sequence of porcine circovirus 2 type primer is following:
Upstream primer P1:5 '-TAA CTA CTC CTC CCG CCA TAC-3 '
Downstream primer P2:5 '-GCC TAC GTG GTC TAC ATT TCC-3 ';
The sequence of pig parvoviral primer is following:
Upstream primer P3:5 '-TGG GAG GGC TTG GTT AGA-3 ';
Downstream primer P4:5 '-TGG TGG TGA GGT TGC TGA T-3 '.
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| CN104450968B (en) * | 2014-12-22 | 2016-10-05 | 福建农林大学 | Primer and probe for pig parvoviral 5 type real time fluorescence quantifying PCR method |
| CN109706267A (en) * | 2019-01-22 | 2019-05-03 | 长江大学 | A kind of universal pig parvoviral nest-type PRC primer sets and its application |
| CN112553317B (en) * | 2019-09-25 | 2022-05-17 | 北京大学人民医院(北京大学第二临床医学院) | Kit for detecting NKG2C genotype and application thereof |
-
2009
- 2009-11-25 CN CN200910172717A patent/CN102071257B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 李明凤等.猪细小病毒SYBR Green Ⅰ实时定量PCR 检测方法的建立.《浙江农业学报》.2009,第21卷(第3期),220-224. * |
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