CN104263852B - Fluorescence quantitative PCR detection pig breathes breeding difficulty disease association virus kit - Google Patents
Fluorescence quantitative PCR detection pig breathes breeding difficulty disease association virus kit Download PDFInfo
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Abstract
The invention discloses the kit that a kind of quadruple real-time fluorescence quantitative PCR detects pig breathing and breeding difficulty disease association virus, comprise 4 pairs of primers of the 100-200bp fragment that can increase, with 4 probes of different fluorophors, be specially: upstream and downstream primer PCV2 virus, upstream and downstream primer PPV virus, upstream and downstream primer CSFV virus, upstream and downstream primer PRRSV virus, specificity PCV2 virus (PRRSV) TaqMan fluorescence probe, specificity PPV virus (PRRSV) TaqMan fluorescence probe, specific C SFV virus (PRRSV) TaqMan fluorescence probe, specificity PRRSV virus (PRRSV) TaqMan fluorescence probe. Adopt the present invention can be quick, accuracy be high, sensitive, and 4 kinds of economic detection pig parvoviral PPV, porcine reproductive and respiratory syndrome virus PRRSV, CSFV CSFV, porcine circovirus 2 type PCV2 etc. cause pig breeding dysfunction disease and porcine respiratory disease virus.
Description
Technical field
The present invention relates to a kind of quadruple and detect porcine circovirus 2 type, pig parvoviral, porcine reproductive and respiratory syndrome virusReal-time fluorescence quantitative PCR kit with CSFV.
Background technology
China's industry of raising pigs develops rapidly at present, and the level difference of raising pigs on different regions, different scales pig farm is very large.Health status and the level of production of swinery are closely bound up. First swine disease shows and causes the straight of pig for the impact of the level of productionConnect death, cause the survival rate of pig to reduce. Even without the death that causes swinery, its health status also can be subject to shadow because of morbidityRing, growing of swinery is obstructed, had a strong impact on the quality of pork pig and boar. Secondly, China's pig industry is just experiencing by faling apartThe type of supporting is to scale, intensive future development, and pig farm scale constantly expands, and the density of raising pigs is concentrated gradually, makes some infectiousnessThe epidemic rate of disease in swinery strengthens, and the extent of injury of swine disease improves day by day, and these have brought all to pig farm epidemic preventing workingChallenge. For a pig farm, once there is deadly infectious disease because epidemic preventing working is not in place, come to the industrial zone of raising pigsHuge loss. Present stage, the principal character of China pig disease transmission was: multiple cause of disease mixed infection, breeding difficulty infectsThe sick ubiquity of sick and breath transmission, and porcine circovirus 2 type, pig parvoviral, porcine reproductive and respiratory syndrome virus and pigThese 4 kinds of viral infection of pestivirus and cross-infection are in the generation of pig breeding dysfunction disease and porcine respiratory disease and play the part of in the grooveDrilling key player.
Pig circular ring virus (Porcinecircovirus, PCV) is a kind of little nonenveloped virus, contains a 1.76kbThe strand cyclic DNA genome of left and right is Circoviridae, Circovirus member. PCV has PCV1 and two kinds of genes of PCV2Type. PCV1 can pollute PK15 clone, is a kind of non-pathogenic virus; PCV2 has pathogenic, can cause the rear multisystem of pig weanThe diseases such as exhaustion syndrome (PMWS), porcine respiratory communicate illness, pig propagating system obstacle. Pig is the natural host of PCV2, withThis disease of easy infection of pig of lactation and finishing period. Epidemiology survey shows, pig circular ring virus is in the swinery of countries in the worldExtensively exist, be easy to clinically breathe and breeding syndrome virus (PRRSV), other diseases such as pig parvoviral (PPV) with pigFormer mixed infection, shows various clinical symptom and pathological change.
Pig parvoviral (Porcineparvovirus, PPV) belongs to Parvoviridae, parvovirus belongs to, its genomeBe a sub-thread minus-strand dna that is about 5000bp, energy self-replacation, comprises two main reading frames. It can cause sowBreeding difficulty, this disease was found in Britain in 1967, and world's every country is all found at present. PPV infects the clinical condition causingShape is embryo and foetal death, sow miscarriage, stillborn foetus and mummy tire etc. At present, the research of PPV is mainly concentrated on to cause of diseaseThe aspects such as, physicochemical property and anti-system, because PRRSV, CSFV etc. also can cause sow breeding difficulty disease, these infectious diseases withPPV infects and is difficult to difference diagnosis, has brought certain difficulty to the control of PPV.
Within 1987, find first pig breeding and breathing syndrome at America & Canada, coming years is popular in Europe respectivelyState. Now this disease extensively exists always and is popular in each country of mainly raising pigs of the world. Clinical manifestation is sow breeding difficulty, presentsThe illnesss such as miscarriage, stillborn foetus, mummy tire and piglet cough, pneumonia. Pig breeding is to be combined by pig breeding and breathing with breathing syndromeClose that syndrome virus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV) causes, rootBe divided into american type (US) and Europe class (EU) separated strain according to science of heredity, clinical symptoms and pathological difference, these two kinds of plant types haveThe homology of 55-70%. At present, the strain great majority that isolated in China obtains are american type.
Swine fever is to belong to flaviviridae, pestivirus by CSFV (Classicalswinefevervirus, CSFV)Belong to, its genome is sub-thread positive chain RNA, is about 12.3kb, contains a large open reading frame (ORF), encodes 3898Amino acid, finally produces 11-12 peptide species. A kind of contagious disease that can cause, causes sow miscarriage, fetus mummy,Deformity, the symptoms such as stillbirth. High pathogenicity rate and the high mortality of swine fever have caused huge economic loss to pig industry, nearly ten yearsGreat changes will take place for its popular and characteristics of incidence, and clinical symptoms lack rule.
Above 4 kinds of cause of diseases all can cause pig reproductive ability obstacle to some extent, and usually mixed infection. Only with clinical conditionShape, pathological change, epidemiology survey are difficult to make Accurate Diagnosis, especially more tired at the early stage of morbidity or diagnosis in incubation periodDifficult. Therefore, set up a kind of high specificity, highly sensitive, reproducible, detection method and the detection that can simultaneously detect multiple cause of diseaseKit is breathed pig and the diagnosis of breeding difficulty disease and prevent and treat significant.
Along with the develop rapidly of various Protocols in Molecular Biologies and the extensive use in medical research thereof, particularly nearestSeveral years rise fluorescent quantitative PCR technique, Real-Time Fluorescent Quantitative PCR Technique in 1996 by U.S. AppliedBiosystemsCompany releases, and because this technology has not only realized the leap of PCR from qualitative to quantitative, and compared with conventional PCR, it has spyThe opposite sex more by force, effectively solves PCR pollution problem, automaticity high, is used widely at present. Therefore, set upAbout porcine circovirus 2 type, pig parvoviral, the multiple fluorescence quantitative of porcine reproductive and respiratory syndrome virus and CSFVThe molecular biology of RT-PCR is quick, and high specificity is highly sensitive, and free of contamination gene tester and diagnostic kit are particularlyImportant.
Summary of the invention
Problem to be solved by this invention is to provide a kind of energy fast, and accuracy is high, sensitive, the economic tiny disease of detection pig4 kinds of poison PPV, porcine reproductive and respiratory syndrome virus PRRSV, CSFV CSFV, porcine circovirus 2 type PCV2 etc. cause that pig is numerousGrow the detection kit of disorder disease and porcine respiratory disease virus.
In order to solve the problems of the technologies described above, to the invention provides a kind of quadruple real-time fluorescence quantitative PCR and detect pig breathing and numerousGrow the kit of disorder disease correlated virus, comprise 4 pairs of primers of the 100-200bp fragment that can increase, with different fluorophors4 probes,
Specific as follows:
One,
Upstream primer PCV2 virus-PR:5 '-CAGCACCCTGTAACGTTTGTC-3 '
Downstream primer PCV2 virus-PR:5 '-TTAGTCTTCCAATCACGCTTCTG-3 '
Specificity PCV2 virus (PRRSV) TaqMan fluorescence probe-P:5 '-HEX-TTTCCCGCTCACGTTCAAAAGTTCAG-BHQ1-3’；
Two,
Upstream primer PPV virus-PR:5 '-GCTTTAGCCTTGGAGCCGT-3 '
Downstream primer PPV virus-PR:5 '-AAGCAGGCTCTTATGTCGGTT-3 '
Specificity PPV virus (PRRSV) TaqMan fluorescence probe-P:5 '-FAM-TGGAGCGAGCCAACAACACCAACTT – BHQ1-3’；
Three,
Upstream primer CSFV virus-PR:5 '-GCTCCCTGGGTGGTCTAAG-3 '
Downstream primer CSFV virus-PR:5 '-CTCGTCCACATAGCATCTCG-3 '
Specific C SFV virus (PRRSV) TaqMan fluorescence probe-P:5 '-ROX-CCTGAGTACAGGACAGTCGTCAGTAGTTCG –BHQ1-3’；
Four,
Upstream primer PRRSV virus-PR:5 '-TCAGCCATAGAAACCTGGAAAT-3 '
Downstream primer PRRSV virus-PR:5 '-ACGACAAATGCGTGGTTATCA-3 '
Specificity PRRSV virus (PRRSV) TaqMan fluorescence probe-P:5 '-TAMRA-ACCTCCAGATGCCGTTTGTGCTTGC –BHQ2-3’。
Detect the reagent of pig breathing and breeding difficulty disease association virus as quadruple real-time fluorescence quantitative PCR of the present inventionThe improvement of box:
Described kit also comprises PCR buffer solution, MgCl2, dNTPs and heat-resisting TaqDNA polymerase, positive reference substance, the moonProperty reference substance, pig parvoviral PPV standard items, porcine reproductive and respiratory syndrome virus PRRSV standard items, CSFV CSFV markAccurate product, pig circular ring virus PCV 2 standard items.
Detect the reagent of pig breathing and breeding difficulty disease association virus as quadruple real-time fluorescence quantitative PCR of the present inventionThe further improvement of box:
4 kinds of viral standard items sequences:
The standard items sequence of PCV2: GAGCACCCTGTAACGTTTGTCAGATTTCCCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACTAA，
The standard items sequence of PPV: GCTTTAGCCTTGGAGCCGTGGAGCGAGCCAACAACACCAACTTTCACCAACCTGCACTTAACTCCAACACCGCCAGATTCAGCAATACGGACACCAAGTCCAACTTGGTCAGAAATAGAAACCGACATAAGAGCCTGCTT，
The standard sequence of CSFV: GCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGACGTGAGCAGAAGCCCACCTCGAGATGCTATGTGGACGAG，
The standard sequence of PRRSV: TCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGGCCGCAAGTACATTCTGGCCCGTGCCCACCACGTTGAAAGTGCCGCAGGCTTTCATCCGATTGCGGCAAATGATAACCACGCATTTGTCGT。
Detect the reagent of pig breathing and breeding difficulty disease association virus as quadruple real-time fluorescence quantitative PCR of the present inventionThe further improvement of box:
Negative control is that the pyrocarbonic acid diethyl ester of autoclave sterilization is processed water, positive control be PCV2, PPV, PRRSV,The positive plasmid sample of CSFV.
Remarks explanations: positive control be the positive plasmid Sample storage of PCV, PPV, PRRSV, CSFV in-20 degree, subtract as far as possibleFew multigelation. , kit of the present invention, is stored in-20 degree, reduces multigelation as far as possible.
The pyrocarbonic acid diethyl ester of autoclave sterilization is processed water (DEPC-H2O) preparation method is: in sterile distilled water, addEnter 0.1% (v/v) pyrocarbonic acid diethyl ester (DEPC), stirring at room temperature is more than 4 hours, and 121 spend autoclavings 20 minutes, cooling standbyWith.
The present invention also provides mentioned reagent box in the application that detects PCV, PPV, PRRSV, CSFV simultaneously.
Particularly: detection kit of the present invention mainly comprises the primer of the 100-200bp fragment that can increase, with differenceThe probe of fluorophor, multi-fluorescence TaqMan probe PCR amplification system reactant liquor, pig parvoviral PPV standard items, pig breedingWith breath syndrome virus PRRSV standard items, CSFV CSFV standard items, porcine circovirus 2 type PCV2 standard items, negative rightAccording to product.
Wherein, multi-fluorescence probe PCR amplification system reactant liquor contains PCR reaction buffer, viral PCV2, PPV,The Auele Specific Primer of PRRSV and CSFV, viral PCV2, PPV, the TaqMan fluorescence probe of PRRSV and CASV;
PCR buffer solution comprises buffer solution, MgCl2, dNTPs and heat-resisting TaqDNA polymerase.
The using method of detection kit of the present invention is specific as follows:
Each detection all should be set up positive control and negative control. Standard items are 1 × 10 with aseptic double-distilled water dilution2～1×106copies/μl。
RNA/DNA viral sample is extracted: according to products instruction, with viral RNA/DNA extraction agent box Ezol(TAKARA) from cell line, fresh or freezing sample extraction viral nucleic acid.
Reverse transcription reaction: the total RNA/DNA mould that adds successively previous step to prepare in the 0.2mlPCR pipe without RNasePlate 0.5 μ L and 1.75 μ LDEPC-H2O, 70 DEG C of sex change 5min, place 3min on ice, add wherein final concentration of inverse transcription reaction liquidFor 1xM-MuLV buffer solution, dNTPs0.1mM, M-MuLV reverse transcriptase 50U, RNase inhibitor 10U, random primer 0.5 μ M,Moisturizing is to cumulative volume 5 μ L. After mixing, at 42 DEG C, react 60min, 70 DEG C of reaction 10min, after reverse transcription reaction finishes, gainedReactant liquor is cDNA/DNA template.
The detection of nucleic acid, for example, can be: get 2 μ L the testing sample nucleic acid RNA/DNA of extracting to be that template is carried out multiple glimmeringLight PCR reaction, comprising: 10 × PCRbuffer2 μ L, 25mMMgCl22.8 μ L, 2.5m Μ dNTP2.9 μ L, institute of the present invention4 pairs of primers stating, i.e. the each 1 μ L of PCV2, PPV, PRRSV and CSFV upstream and downstream primer (10 μ M), PCV2 of the present invention, PPV,The each 0.5 μ L of tetra-kinds of probes of PRRSV and CSFV (10 μ M), TaqDNAPolymerase0.2U, adds ddH2O is total to reaction systemVolume is 20 μ L. Response parameter is: 95 DEG C of 3min; 95 DEG C of 15s, 60 DEG C of 1min, totally 40 circulations.
Fluorescence PCR system for example can be set to:
Kit: multiple fluorescence quantitative PCR reaction system (total reaction volume 50 μ L): four kinds of standard items templates (1 ×106copies/μL)4μL，10×PCRbuffer5μL，25mMMgCl25μL，dNTP(10mΜ)1.5μL，TaqDNAPolymerase2.5ul, the each 2 μ L of four kinds of primers (10mM/L), four kinds of probes (10mM/L), 1 μ L, supplies ddH2O to 50 μ L.Establish a negative control (is: the pyrocarbonic acid diethyl ester that cDNA template is made into autoclave sterilization is processed water, and volume is not simultaneouslyBecome). Reaction is carried out on ABI7300 fluorescent quantitation instrument, and amplification program is 95 DEG C of 5min; 40 circulations: 95 DEG C of 30s, 56 DEG C30s, 72 DEG C of 31s. Result judgement: select fluoroscopic examination model F AM, HEX, ROX, the adjustment of CY5 fluorescence baseline to get 3-15 circulationFluorescence signal mean value, Threshold is the peak just above normal negative control product with threshold line, it is typical that sample isAmplification curve, is judged as the positive. Without typical amplification curve, be judged as feminine gender.
Fluorescent quantitation report the test:
1) the amplification curve Ct of PCV2, PPV, PRRSV, CSFV (thresholdcycle, the fluorescence in each reaction tubeThe amplification cycles number that signal experiences while reaching set threshold value) be equal to 40.0, be judged to ' negative ' specimens.
2) if the sample of Ct value≤35 of PCV2, PPV, PRRSV, the corresponding amplification curve of CSFV wherein, testing resultPositive corresponding to this kind of virus, testing sample carries corresponding virus;
3) Ct value is gray area in 35～40 intervals, after reforming, is greater than 37, is judged to feminine gender.
4), if testing result is positive, according to obtained calibration curve and testing sample CT value, calculate sample to be measuredVirus copy number/ml.
Adopt technique scheme, the technological progress of this kit is: (1) has been designed and point once can have been detected 4 kinds of pigsThe probe that the multiple real time fluorescence PCR of important virus detects, has overcome existing molecular detecting method complicated operation, and sensitivity is notHeight, specificity is not strong, the repeatability shortcoming such as bad. (2) multiple real time fluorescence PCR detection method of the present invention can detect 4 of pigPlant important virus disease, and can detect a large amount of samples by this kit. (3) the present invention not only can qualitative detectionPCV2, PPV, PRRSV, CSFV, can also judge the content of various virus in sample by analytical standard curve. (4) originallyInvention has very high sensitivity, the minimum correlated virus that can detect 10 copy numbers in sample. (5) the present invention detects soonSpeed, high specificity, except probe, all the other reagent are all regular-PCR products of domestic biotech firm, are improving swine disease poison detection skillWhen art level, also greatly save cost.
Brief description of the drawings
Fig. 1 is the structural representation of kit.
Fig. 2 is the example that multiple fluorescence quantitative PCR system detects.
Fig. 3 is the sensitivity of fluorescence quantitative PCR detection porcine circovirus 2 type, is respectively 10 from left to right6、105、104、103、102、10copies/μL。
Fig. 4 is the sensitivity of fluorescence quantitative PCR detection pig parvoviral, is respectively 10 from left to right6、105、104、103、102、10copies/μL。
Fig. 5 is the sensitivity of fluorescence quantitative PCR detection porcine reproductive and respiratory syndrome virus, is respectively 10 from left to right6、105、104、103、102、10copies/μL。
Fig. 6 is the sensitivity of fluorescence quantitative PCR detection CSFV, is respectively 10 from left to right6、105、104、103、102、10copies/μL。
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is further described, but to protection scope of the present invention alsoBe not limited only to this.
Embodiment 1,
Referring to Fig. 1, a kind of quadruple real-time fluorescence quantitative PCR detection kit, by quantitative PCR reactant liquor 1, pig parvoviralPPV standard items 2, porcine reproductive and respiratory syndrome virus PRRSV standard items 3, CSFV CSFV standard items 4,2 porcine circovirusType PCV2 standard items 5, positive reference substance 6, negative control product 7, box body 8 form.
Box body 8 is provided with container hole, places respectively quantitative PCR reactant liquor 1, the breeding of pig parvoviral PPV standard items 2, pig withBreath syndrome virus PRRSV standard items 3, CSFV CSFV standard items 4, porcine circovirus 2 type PCV2 standard items 5, the positiveReference substance 6, negative control product 7; Wherein quantitative PCR reactant liquor single tube packing, matrix is arranged, and fluorescence quantitative PCR reaction solution containsPCR reaction buffer (chloride containing manganese and triphosphate deoxyribose nucleotide mixture, heat-resisting TaqDNA polymerase etc.) 4 pipe (marksBe designated as ★), four kinds of viral Auele Specific Primers (upstream and downstream primer is with pipe dress) are 4 pipes (being labeled as ◆), corresponding four kinds of fluorescenceProbe is 4 pipes (being labeled as ▲).
Embodiment 2,
1 test material and method
1.1 strain pig source circovirus virus 2 types, pig parvoviral, porcine reproductive and respiratory syndrome virus, CSFV eachStrain.
1.2 samples to be checked---serum collection, from test pig, gathers from the pig farm of Fujian, Shanghai, zhejiang and other places respectively;
1.3 primers and probe
From the NCBI gene pool of the U.S. downloaded porcine circovirus 2 type all over the world, pig parvoviral, pig breeding withBreath syndrome virus, CSFV gene order. It is carried out to homology comparison, at the virus genomic conservative base of correspondenceBecause of district's design Auele Specific Primer and TaqMan probe, sequence is as follows:
One,
Upstream primer PCV2 virus-PR:5 '-CAGCACCCTGTAACGTTTGTC-3 '
Downstream primer PCV2 virus-PR:5 '-TTAGTCTTCCAATCACGCTTCTG-3 '
Specificity PCV2 virus (PRRSV) TaqMan fluorescence probe-P:5 '-HEX-TTTCCCGCTCACGTTCAAAAGTTCAG-BHQ1-3’；
Two,
Upstream primer PPV virus-PR:5 '-GCTTTAGCCTTGGAGCCGT-3 '
Downstream primer PPV virus-PR:5 '-AAGCAGGCTCTTATGTCGGTT-3 '
Specificity PPV virus (PRRSV) TaqMan fluorescence probe-P:5 '-FAM-TGGAGCGAGCCAACAACACCAACTT – BHQ1-3’；
Three,
Upstream primer CSFV virus-PR:5 '-GCTCCCTGGGTGGTCTAAG-3 '
Downstream primer CSFV virus-PR:5 '-CTCGTCCACATAGCATCTCG-3 '
Specific C SFV virus (PRRSV) TaqMan fluorescence probe-P:5 '-ROX-CCTGAGTACAGGACAGTCGTCAGTAGTTCG –BHQ1-3’；
Four,
Upstream primer PRRSV virus-PR:5 '-TCAGCCATAGAAACCTGGAAAT-3 '
Downstream primer PRRSV virus-PR:5 '-ACGACAAATGCGTGGTTATCA-3 '
Specificity PRRSV virus (PRRSV) TaqMan fluorescence probe-P:5 '-TAMRA-ACCTCCAGATGCCGTTTGTGCTTGC –BHQ2-3’。
Primer and probe entrust Sangon Biotech (Shanghai) Co., Ltd. synthetic.
The extraction of 1.4 viral DNA quantitative criterions and viral nucleotide:
RNA/DNA viral sample is extracted: according to products instruction, with viral RNA/DNA extraction agent box Ezol(TAKARA) from cell line, fresh or freezing sample extraction viral nucleic acid.
Reverse transcription reaction: the total RNA template 0.5 that adds successively previous step to prepare in the 0.2mlPCR pipe without RNaseμ L and 1.75 μ LDEPC-H2O, 70 DEG C of sex change 5min, place 3min on ice, and (final concentration is 1 × M-to add inverse transcription reaction liquidMuLV buffer solution, dNTPs0.1mM, M-MuLV reverse transcriptase 50U, RNase inhibitor 10U, random primer 0.5uM, moisturizing is extremelyCumulative volume 5 μ L). After mixing, at 42 DEG C, react 60min, 70 DEG C of reaction 10min, after reverse transcription reaction finishes, gained reactionLiquid is cDNA/DNA template.
1.5 Fluorescence PCR systems:
Kit: multiple fluorescence quantitative PCR reaction system (total reaction volume 50 μ L): four kinds of standard items templates (1 ×106copies/μL)4μL，10×PCRbuffer5μL，25mMMgCl25μL，dNTP(10mΜ)1.5μL，TaqDNAPolymerase2.5ul, the each 2 μ L of four kinds of primers (10mM/L), four kinds of probes (10mM/L), 1 μ L, supplies ddH2O to 50 μ L.Establish a negative control (is: the pyrocarbonic acid diethyl ester that cDNA template is made into autoclave sterilization is processed water, and volume is not simultaneouslyBecome). Reaction is carried out on ABI7300 fluorescent quantitation instrument, and amplification program is 95 DEG C of 5min; 40 circulations: 95 DEG C of 30s, 56 DEG C30s, 72 DEG C of 31s. Result judgement: select fluoroscopic examination model F AM, HEX, ROX, the adjustment of CY5 fluorescence baseline to get 3-15 circulationFluorescence signal mean value, Threshold is the peak just above normal negative control product with threshold line, it is typical that sample isAmplification curve, is judged as the positive. Without typical amplification curve, be judged as feminine gender.
1.6 fluorescent PCR specificitys, sensitivity and replica test
Select porcine circovirus 2 type, pig parvoviral, the positive of porcine reproductive and respiratory syndrome virus and CSFVThe pig serum sample of cDNA and collection, extracts nucleic acid to above-mentioned clinical sample, and with porcine circovirus 2 type, pig parvoviral, pig is numerousGrow with breath syndrome virus and CSFV multiple fluorescence PCR method and detect, the specificity of verification method; To demarcatingThe porcine circovirus 2 type (1 × 10 of copy number (copies/ μ L)6Copies/ μ L), pig parvoviral (1 × 106copies/μL)，Porcine reproductive and respiratory syndrome virus and CSFV (1 × 106Copies/ μ L) and CSFV (1 × 106Copies/ μ L) pointNot Xi Shi after, the parallel Fluorescence PCR that carries out, relatively its sensitivity. In addition, the viral nucleic acid dilution of each concentration is done to 3Inferior duplicate detection, the Ct value obtaining is calculated standard deviation, the repeatability of verification method.
2 results
2.1 specificity
The multiple fluorescence PCR method that the present invention sets up is to porcine circovirus 2 type, pig parvoviral, pig breeding and breathe comprehensiveSimulator sickness virus and CSFV have good specificity, and the sample of collection also detects positive reaction. And with other virus asSwine influenza virus, bovine viral diarrhea virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus etc. are all anti-without intersectingShould. Result is referring to Fig. 2, and the amplification curve that in figure, A is porcine circovirus 2 type, amplification curve, the C that B is pig parvoviral are that pig is numerousGrow and the amplification curve of breath syndrome virus, the amplification curve that D is CSFV, and viral as swine flu disease to remainingPoison, bovine viral diarrhea virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and negative control are all without significantly expandingIncrease curve.
2.2 sensitivity
Respectively to porcine circovirus 2 type, pig parvoviral, porcine reproductive and respiratory syndrome virus, CSFV dilution(, concentration is respectively 106、105、104、103、102, 10copies/ μ L) after, detect result fluorescence with fluorescence PCR methodPCR method detection sensitivity reaches respectively 10,10,10,10copies/ μ L (seeing Fig. 3,4,5,6).
2.3 repeated
Get respectively porcine circovirus 2 type (1 × 106Copies/ μ L), pig parvoviral (1 × 106Copies/ μ L), pigReproductive and respiratory syndrome virus (1 × 106Copies/ μ L), CSFV dilution (1 × 106Copies/ μ L) mix afterwards by 10Doubly be diluted to 3 different concentration, repeat in the sample of each concentration is done to 3 batches and batch between repeat, the different nucleic acid of result is denseSpend various detection Ct value standard deviation≤0.2 and≤0.5. There is good repeatability (table 1).
Table 1 fluorescence RT-PCR detects the replica test of swine disease poison
2.4 clinical samples detect
By gathering from Zhejiang, 60 parts of the pig serum samples on the ground such as Fujian, Shanghai, directly extract nucleic acid, with of the present inventionFluorescence PCR method detects virus, adopts conventional method to detect simultaneously and does parallel test.
Regular-PCR method detects with fluorescence quantifying PCR method testing result of the present invention as follows:
PCV2 regular-PCR detects positive 11 parts, and the present invention detects positive 13 parts;
PPV regular-PCR detects positive 0 part, and the present invention detects positive 1 part;
PRRSV regular-PCR detects positive 17 parts, and the present invention detects positive 18 parts;
CSFV regular-PCR detects positive 3 parts, and the present invention detects positive 3 parts;
Wherein 1 part of CSFV and PRRSV mixed infection, 8 parts of PRRSV and PCV2 mixed infections.
Remarks explanation: detect and learn according to the most authoritative detection method substance real-time fluorescence quantitative PCR of the industry: it detects knotFruit is with the testing result of gained of the present invention.
It is high that PCR fluorescent method of the present invention relatively obtains positive rate for the ratio conventional method of pattern detection, and can be simultaneouslyDetect multiple infection, simple to operate, safety automation degree is high, and detection speed is fast, adds extraction and the reverse transcription of nucleic acid, altogether onlyNeed 3-4 hour, with the obvious advantage.
Comparative example, the probe of PCV2 and PPV is done to following change:
Specificity PCV2 virus (PRRSV) TaqMan fluorescence probe-P:5 '-HEX-TTTCCCGCTCACTTTCAAAAGTTCAGC-BHQ1-3’
Specificity PPV virus (PRRSV) TaqMan fluorescence probe-P:5 '-FAM-AGCGAGCCAACAACACCAACTTTCAC –BHQ1-3’；
All the other are with above-described embodiment 2.
The result of sensitivity test is: porcine circovirus 2 type, pig parvoviral, porcine reproductive and respiratory syndrome virusSensitivity reaches respectively 80,80,80,80copies/mL.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention. Obviously, thisBrightly be not limited to above embodiment, can also have many distortion. Those of ordinary skill in the art can be from content disclosed by the inventionAll distortion of directly deriving or associating, all should think protection scope of the present invention.
<110>Institutes Of Technology Of Zhejiang
<120>fluorescence quantitative PCR detection pig breathes breeding difficulty disease association virus kit
<160>12
<210>1
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>upstream primer PCV2 virus
<400>1
cagcaccctgtaacgtttgtc21
<210>2
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>downstream primer PCV2 virus
<400>2
ttagtcttccaatcacgcttctg23
<210>3
<211>26
<212>DNA
<213>artificial sequence
<220>
<223>specificity PCV2 virus (PRRSV) TaqMan fluorescence probe
<400>3
tttcccgctcacgttcaaaagttcag26
<210>4
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>upstream primer PPV virus
<400>4
gctttagccttggagccgt19
<210>5
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>downstream primer PPV virus
<400>5
aagcaggctcttatgtcggtt21
<210>6
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>specificity PPV virus (PRRSV) TaqMan fluorescence probe
<400>6
tggagcgagccaacaacaccaactt25
<210>7
<211>19
<212>DNA
<213>artificial sequence
<220>
<223>upstream primer CSFV virus
<400>7
gctccctgggtggtctaag19
<210>8
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>downstream primer CSFV virus
<400>8
ctcgtccacatagcatctcg20
<210>9
<211>30
<212>DNA
<213>artificial sequence
<220>
<223>specific C SFV virus (PRRSV) TaqMan fluorescence probe
<400>9
cctgagtacaggacagtcgtcagtagttcg30
<210>10
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>upstream primer PRRSV virus
<400>10
tcagccatagaaacctggaaat22
<210>11
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>downstream primer PRRSV virus--
<400>11
acgacaaatgcgtggttatca21
<210>12
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>specificity PRRSV virus (PRRSV) TaqMan fluorescence probe
<400>12
acctccagatgccgtttgtgcttgc25
Claims (3)
1. quadruple real-time fluorescence quantitative PCR detects the kit of pig breathing and breeding difficulty disease association virus, it is characterized in that: bagDraw together 4 pairs of primers of the 100-200bp fragment that can increase, with 4 probes of different fluorophors,
Specific as follows: one,
Upstream primer PCV2 virus-PR:5 '-CAGCACCCTGTAACGTTTGTC-3 '
Downstream primer PCV2 virus-PR:5 '-TTAGTCTTCCAATCACGCTTCTG-3 '
Specificity PCV2 virus (PRRSV) TaqMan fluorescence probe-P:5 '-HEX-TTTCCCGCTCACGTTCAAAAGTTCAG-BHQ1-3 ';
Two,
Upstream primer PPV virus-PR:5 '-GCTTTAGCCTTGGAGCCGT-3 '
Downstream primer PPV virus-PR:5 '-AAGCAGGCTCTTATGTCGGTT-3 '
Specificity PPV virus (PRRSV) TaqMan fluorescence probe-P:5 '-FAM-TGGAGCGAGCCAACAACACCAACTT – BHQ1-3 ';
Three,
Upstream primer CSFV virus-PR:5 '-GCTCCCTGGGTGGTCTAAG-3 '
Downstream primer CSFV virus-PR:5 '-CTCGTCCACATAGCATCTCG-3 '
Specific C SFV virus (PRRSV) TaqMan fluorescence probe-P:5 '-ROX-CCTGAGTACAGGACAGTCGTCAGTAGTTCG –BHQ1-3’；
Four,
Upstream primer PRRSV virus-PR:5 '-TCAGCCATAGAAACCTGGAAAT-3 '
Downstream primer PRRSV virus-PR:5 '-ACGACAAATGCGTGGTTATCA-3 '
Specificity PRRSV virus (PRRSV) TaqMan fluorescence probe-P:5 '-TAMRA-ACCTCCAGATGCCGTTTGTGCTTGC – BHQ2-3’。
2. quadruple real-time fluorescence quantitative PCR according to claim 1 detects pig breathing and breeding difficulty disease association virusKit, it is characterized in that:
Described kit also comprises PCR buffer solution, MgCl2, dNTPs and heat-resisting TaqDNA polymerase, positive reference substance, negative rightAccording to product, pig parvoviral PPV standard items, porcine reproductive and respiratory syndrome virus PRRSV standard items, CSFV CSFV standardProduct, pig circular ring virus PCV 2 standard items;
Negative control product are that the pyrocarbonic acid diethyl ester of autoclave sterilization is processed water, positive reference substance be PCV2, PPV, PRRSV andThe positive plasmid sample of CSFV.
3. quadruple real-time fluorescence quantitative PCR according to claim 2 detects pig breathing and breeding difficulty disease association virusKit, it is characterized in that:
4 kinds of viral standard items sequences:
The standard items sequence of PCV2: GAGCACCCTGTAACGTTTGTCAGATTTCCCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACTAA，
The standard items sequence of PPV: GCTTTAGCCTTGGAGCCGTGGAGCGAGCCAACAACACCAACTTTCACCAACCTGCA CTTAACTCCAACACCGCCAGATTCAGCAATACGGACACCAAGTCCAACTTGGTCAGAAATAGAAACCGACATAAGAGCCTGCTT，
The standard sequence of CSFV: GCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGACGTGA GCAGAAGCCCACCTCGAGATGCTATGTGGACGAG，
The standard sequence of PRRSV: TCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGG CCGCAAGTACATTCTGGCCCGTGCCCACCACGTTGAAAGTGCCGCAGGCTTTCATCCGATTGCGGCAAATGATAACCACGCATTTGTCGT。
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| CN107974516A (en) * | 2017-12-22 | 2018-05-01 | 山东新希望六和集团有限公司 | For detecting primer and probe, the PCR kit for fluorescence quantitative and methods and applications of porcine circovirus 2 type |
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| CN107267664A (en) * | 2017-05-19 | 2017-10-20 | 河南大学 | A kind of ddPCR methods, primer and its application and kit that porcine reproductive and respiratory syndrome virus is detected for absolute quantitation |
| CN108384890A (en) * | 2018-03-29 | 2018-08-10 | 河南省动物疫病预防控制中心 | Differentiate the double FQ-PCR detection kits of porcine circovirus 2 type and 3 types |
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| CN103509882A (en) * | 2013-06-28 | 2014-01-15 | 广东温氏食品集团股份有限公司 | Fluorescent quantitative PCR primers and probes for porcine epidemic diarrhea viruses |
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| 多重PCR同时检测猪圆环病毒2 型, 猪细小病毒, 猪繁殖与呼吸综合征;姜永厚;《中国兽医学报》;20091031;第29卷(第10期);1237-1241 * |
| 鉴别PCV2、PPV、PRV、PRRSV、CSFV多重PCR诊断方法的建立及初步应用;岳丰雄;《中国优秀硕士学位论文全文数据库 农业科技辑》;20081015(第10期);摘要和第17-18页 * |
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| CN107974516A (en) * | 2017-12-22 | 2018-05-01 | 山东新希望六和集团有限公司 | For detecting primer and probe, the PCR kit for fluorescence quantitative and methods and applications of porcine circovirus 2 type |
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