CN104531897A - Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) - Google Patents
Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) Download PDFInfo
- Publication number
- CN104531897A CN104531897A CN201410742262.5A CN201410742262A CN104531897A CN 104531897 A CN104531897 A CN 104531897A CN 201410742262 A CN201410742262 A CN 201410742262A CN 104531897 A CN104531897 A CN 104531897A
- Authority
- CN
- China
- Prior art keywords
- prrsv
- csfv
- seq
- quantitative pcr
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M). The method realizes simultaneous detection of existence of CSFV, PRRSV-U and PRRSV-M in a sample to be detected and can realize quantification of loading levels of all types of viruses. The invention also provides the kit for three-color fluorescent quantitative PCR combined detection of CSFV, PRRSV-U and PRRSV-M. The method and the kit have the advantages of operation simpleness and fastness, high singularity, sensitive and reliable detection effects, and lowest detection concentration of 1*10<2> cps per microliter.
Description
Technical field
The present invention relates to the universal and detection method that highly pathogenic PRRS is malicious of a kind of Pestivirus suis, pig blue-ear disease poison, be specifically related to a kind ofly detect Pestivirus suis, pig blue-ear disease poison is universal and highly pathogenic PRRS is malicious three fluorescence quantitative PCR associated detecting method and detection kit thereof simultaneously.
Background technology
Swine fever is a kind of high degree in contact sexually transmitted disease hot in nature caused by Pestivirus suis (Classical Swine Feine Virus, CSFV), and this disease transmission is high, by force pathogenic, and pig is the unique natural reservoir (of bird flu viruses) of this disease.The sick pig main manifestations infected is characteristic pathological change, visceral hemorrhage, and infraction and necrosis, mortality ratio is very high, has carried out great financial loss to industrial belt of raising pigs.Sick pig is this sick topmost contagium, and it is the major way of virus disseminating that susceptible pig and sick pig direct contacts.In China, swine fever is popular presents classical swine fever and non-typical swine fever coexists, persistent infection and stealthyly infect forms such as coexisting, immunological tolerance and band poison syndrome coexist; Abroad, the swine fever of Belgium, Germany, Holland's outburst also shows, the popular form of swine fever is changing, and this brings new challenge to global pig industry.CSFV and bovine viral diarrhea virus (Bivine viral disease virus, and sheep border disease virus (border disease virus BVDV), BDV) flaviviridae (Flaviviridae) pestivirus (pestivirus) is belonged to together, when carrying out Detection of antigen, cross-immune reaction can be there is, and for special primer, the probe of gene design, then can effectively avoid intersection to detect.Pestivirus suis genome is single-stranded positive RNA, is about 12.3KB, and containing a large open reading frame (ORF), virus particle is spherical in shape, diameter 40-50nm, icosahedral symmetry, and its core marrow diameter 29-30nm, has cyst membrane.
Pig blue-ear disease and porcine reproductive and respiratory syndrome, by PRRS virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) a kind of high degree in contact sexually transmitted disease caused, in epidemic infection in most countries.Clinical manifestation very different after pig morbidity is one of notable feature of this disease.Clinical symptom is breeding difficulty mainly, comprises miscarriage later stage of pregnancy, premature labor and stillborn foetus, the respiratory symptom of bred pigs.Morbidity sow fervescence, apocleisis, the dorsal part of part affected pig, the have sharp ears of ears and edge are reddish blue.The hyposexuality of morbidity boar, production performance declines.Individual little after dead or output during the birth of morbidity piglet.Main pathological change after affected pig death is: eyeball swelling is given prominence to, head, buttocks subcutaneous dropsy, thoracic cavity, hydropericardium, ventricular dilatation, and cardiac muscle change atrophy, Renal Cortex portion petechial hemorrhage, lungs are interstitial pneumonia pathology.Cause of disease first identified in 1991, virus is single strand RNA virus, belongs to Arteriviridae Arterivirus, but the source of virus is not determined.The existence of U.S.'s swinery porcine reproductive and respiratory syndrome virus serum antibody in 1985 illustrates that this virus just existed before morbidity.
Highly pathogenic PRRS is by porcine reproductive and respiratory syndrome (PorcineReproductive and Respiratory Syndrom, PRRS) the acute high lethality epidemic disease that causes of virus variant, it is current popular wide, transmissible disease that harm is very serious, two class transmissible diseases are decided to be at present by China, because it can cause the high mortality of sow breeding difficulty, piglet and growing and fattening pigs, especially morbidity pig immunosuppression dysfunction can be caused, therefore huge financial loss can be caused to pig industry.At present.Detecting the method for PRRSV infection has multiple, and comprise virus purification, Detection of antigen and antibody test etc., the operation that these methods have is more loaded down with trivial details, have with certain subjectivity, accuracy is not high.
PRRSV is divided into 2 genotype, i.e. gene 1 type and gene 2 type, and the former is with Lelystad virus(LV strain) be the Europe class strain of representative, the North America type strain that the latter is is representative with ATCC VR-2332 strain.PRRSV has the diversity of variability and strain widely, and there is significant antigenic diversity between Europe and North America isolated strain, both only have very weak cross reaction, different strain virulence and pathogenicly also have obvious difference.The PRRSV isolated strain of China belongs to gene 2 type mostly, i.e. North America type, has been reported recently and isolated Europe class strain from clinical samples.Also there is the diversity of variation phenomenon and strain widely in the isolated strain of China.Within 2002, be separated to NS2 Protein (Nsp2) coding region consecutive miss 12 amino acid whose strains in the world first, the highly pathogenic strain occurred for 2006 is with the discontinuous disappearance in Nsp2 coding region 30 amino acid (481 disappearances, 1 amino acid, 533-561 consecutive miss 29 amino acid) for feature, and lethality is presented to pig.
Pestivirus suis, universal and that highly pathogenic PRRS the is malicious detection method of pig blue-ear disease poison mainly comprise immunological detection method and the large class of nucleic acid detection method two in short.Immunological method mainly detects the specific antibody whether depositing swine fever and PRRS virus in serum, specifically detect by the method for ELISA, but this method must be based upon virus infection occur and produce on the basis of antibody, and in view of the variability of above-mentioned virus and the intercrossing with other virus serology, its specificity is difficult to ensure, easily occurs false positive and false negative.Virus purification and culture method more responsive, but this method complex operation, should not all test experience particularly some laboratories promote.Swine fever or PRRS virus nucleic acid is carried out by the method for regular-PCR, although sensitivity increases, but easily produce non-specific amplification, even can there is false positive because of the reason of operation, and the judgement of its result of regular-PCR TRAP needs to carry out gel electrophoresis analysis to product, working method simplifies not.
Fluorescent quantitative PCR technique, refers to and add fluorophor in PCR reaction system, and utilize fluorescent signal to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis, the method is convenient to operation.CN101058830A discloses " pig pestivirus fluorescence quantitative RT-PCR diagnosis agent kit ", and CN101328506A discloses " fluorescent quantitative PCR rapid diagnosis reagent box that a species specificdetection wild-type classical swine fever virus infects and application method thereof ".Two sections of documents have all only carried out independent detection to Pestivirus suis.Universal and the highly pathogenic PRRS of swine fever, pig blue-ear disease poison is malicious is the virus disease of serious harm pig, and often with mode infected pigs that is independent or polyinfection, M & M is all higher.At present also imperfect for Pestivirus suis, pig blue-ear disease poison associated detecting method that is universal and the malicious infection of highly pathogenic PRRS.
Multicolor fluorescence quantitative PCR technique refers to multiple goal gene that simultaneously to increase in the test of same fluorescent quantitative PCR, and the fluorescent probe without wavelength that each goal gene adopts detects.Fluorescently-labeled probe has multiple, such as FAM, VIC, JOE, NED, HEX etc., the fluorescent signal difference that often kind of fluorescently-labeled probe produces in pcr amplification process.
Summary of the invention
The invention provides a kind of Pestivirus suis, PRRS virus is universal and highly pathogenic PRRS is malicious three fluorescence quantitative PCR associated detecting method, the method can be implemented in same sample to be tested the existence simultaneously detecting Pestivirus suis (CSFV), PRRS virus (PRRSV-U) and high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M), and can to detect virus load level carry out quantitatively.Present invention also offers a kind of test kit for Pestivirus suis, PRRS virus (classic and high pathotype is general), high-pathogenicity porcine reproductive and respiratory syndrome virus three fluorescence quantitative PCR joint-detection.
The technical solution used in the present invention is:
Pestivirus suis of the present invention (CSFV), PRRS virus (classic and high pathotype is general) (PRRSV-U), high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M) three fluorescence quantitative PCR, comprise the steps:
1) viral RNA in testing sample is extracted;
2) with the total serum IgE obtained for template, use Pestivirus suis (CSFV)
Upstream primer: CSFV-F:5 '-CAGTAGTTCGACGTGAGCAGAAG-3 ' (SEQ ID NO.1);
Downstream primer: CSFV-R:5 '-CGCTAGGGTTAAGGTGTGTCTTG-3 ' (SEQ ID NO.2);
Fluorescent probe: CSFV-P:5 '-ACCTCGAGATGCTACGTGGACGA-3 ' (SEQ ID NO.3)
Universal with PRRS virus:
Upstream primer: PRRSV-UF:5 '-GGTTGGCTTTTGCTGTTGGT-3 ' (SEQ ID NO.4);
Downstream primer: PRRSV-UR:5 '-TCTGGCGAGTCAAACTCACAA-3 ' (SEQ ID NO.5);
Fluorescent probe: PRRSV-UP:5 '-VIC-TGTTCAAGCCTGTGTCCGACCCAG-BHQ1-3 ' (SEQ ID NO.6).
With high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M)
Upstream primer: PRRSV-MF:5 '-AGCTGATGACACCTTTGAGTGAGT-3 ' (SEQ ID NO.7);
Downstream primer: PRRSV-MR:5 '-GACAAATCCAGAGGCTCATCCT-3 ' (SEQ ID NO.8),
Fluorescent probe: PRRSV-MP:5 '-CY5-AGAACTGTGACAACAACGCTGACGCAC-BHQ2-3 ' (SEQ ID NO.9)
Carry out three fluorescence quantitative PCR detection, wherein, Pestivirus suis, PRRS virus are universal, the fluorescent probe reporter group of high-pathogenicity porcine reproductive and respiratory syndrome virus is not identical;
3) according to the fluorescence Ct value of testing sample, interpretation of result: after reaction terminates, judges whether testing sample is that CSFV, PRRSV-U, PRRSV-M are positive.
The reaction system cumulative volume of described quantitative fluorescent PCR reaction is 25 μ l, composed as follows: the swine fever that multicolor fluorescence quantitative PCR MIX 20 μ l, ThermoScript II are 0.5 μ l, Taq enzyme is 0.5 μ l and extraction and PRRS virus RNA or positive quality control product or negative quality control product 4 μ l; Wherein contain 10 × multicolor fluorescence quantitative PCR buffer, dNTPs in three fluorescence quantitative PCR MIX.
Containing following composition: 500mMTris-HCl (pH8.0), 30mM MgCl in described 10 × multicolor fluorescence quantitative PCR buffer
2, 250mM KCl and 15% (v/v) the RNaseOut (U) of DMSO, 10U.
A preferred version of described detection method is that multicolor fluorescence quantitative PCR MIX consists of:
10 × multicolor fluorescence quantitative PCR buffer 2.5 μ l
CSFV-F（10μM） 1μl
CSFV-R（10μM） 1μl
PRRSV-UF（10μM） 1μl
PRRSV-UR（10μM） 1μl
PRRSV-MF（10μM） 1μl
PRRSV-MR（10μM） 1μl
CSFV-P（10μM） 0.5μl
PRRSV-UP（10μM） 0.5μl
PRRSV-MP（10μM） 0.5μl
dNTPS (10mM) 1μl
ddH
2O 1.5μl
Total 20.0μl。
A preferred version of described detection method is that multicolor fluorescence quantitative PCR reaction conditions is: 93 DEG C 2 minutes, then 93 DEG C 30 seconds, 55 ~ 58 DEG C 45 seconds, gather fluorescent signal, 40 circulations.
One detects Pestivirus suis simultaneously, the three fluorescence quantitative PCR combined detection kit that pig blue-ear disease poison is universal and highly pathogenic PRRS is malicious, comprise following composition: viral nucleic acid extracting solution A, viral nucleic acid extracting solution B, ThermoScript II system, Taq enzyme system, multicolor fluorescence quantitative PCR MIX, positive quality control product, negative quality control product, containing 10 × multicolor fluorescence quantitative PCR buffer in described multicolor fluorescence quantitative PCR MIX, dNTPS, and Pestivirus suis, PRRS virus is universal, the Auele Specific Primer of high-pathogenicity porcine reproductive and respiratory syndrome virus and polychromatic probe, sequence is respectively:
Pestivirus suis:
Upstream primer: CSFV-F:5 '-CAGTAGTTCGACGTGAGCAGAAG-3 ' (SEQ ID NO.1);
Downstream primer: CSFV-R:5 '-CGCTAGGGTTAAGGTGTGTCTTG-3 ' (SEQ ID NO.2);
Fluorescent probe: CSFV-P:5 ' 6-Fam-ACCTCGAGATGCTACGTGGACGA-BHQ1-3 ' (SEQ ID NO.3)
PRRS virus is universal:
Upstream primer: PRRSV-UF:5 '-GGTTGGCTTTTGCTGTTGGT-3 ' (SEQ ID NO.4);
Downstream primer: PRRSV-UR:5 '-TCTGGCGAGTCAAACTCACAA-3 ' (SEQ ID NO.5);
Fluorescent probe: PRRSV-UP:5 '-VIC-TGTTCAAGCCTGTGTCCGACCCAG-BHQ1-3 ' (SEQ ID NO.6).
High-pathogenicity porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-MF:5 '-AGCTGATGACACCTTTGAGTGAGT-3 ' (SEQ ID NO.7);
Downstream primer: PRRSV-MR:5 '-GACAAATCCAGAGGCTCATCCT-3 ' (SEQ ID NO.8),
Fluorescent probe: PRRSV-MP:5 '-CY5-AGAACTGTGACAACAACGCTGACGCAC-BHQ2-3 ' (SEQ ID NO.9)
Wherein, the fluorescent probe reporter group of Pestivirus suis is FAM, and quenching group is BHQ1; The universal fluorescent probe reporter group of PRRS virus is VIC, and quenching group is BHQ1; The fluorescent probe reporter group of high-pathogenicity porcine reproductive and respiratory syndrome virus is CY5, and quenching group is BHQ2.
A preferred version of described detection kit is, containing following composition: 500mMTris-HCl (pH8.0), 30mM MgCl in 10 × multicolor fluorescence quantitative PCR buffer
2, 250mM KCl and 15% (v/v) the RNaseOut (U) of DMSO, 10U.
A preferred version of described detection kit is that multicolor fluorescence quantitative PCR MIX's is composed as follows:
10 × multicolor fluorescence quantitative PCR buffer 2.5 μ l
CSFV-F（10μM） 1μl
CSFV-R（10μM） 1μl
PRRSV-UF（10μM） 1μl
PRRSV-UR（10μM） 1μl
PRRSV-MF（10μM） 1μl
PRRSV-MR（10μM） 1μl
CSFV-P（10μM） 0.5μl
PRRSV-UP（10μM） 0.5μl
PRRSV-MP（10μM） 0.5μl
dNTPS (10mM) 1μl
ddH
2O 1.5μl
Total 20.0μl。
A preferred version of described detection kit is, contain in the every 1000ml of viral extract A: guanidinium isothiocyanate 480g, 0.1M Tris-HCl(pH 6.4) 500ml, 0.2M EDTA(pH8.0) 120ml, surplus is water, and viral extract B consists of Virahol.
The invention has the beneficial effects as follows:
The inventive method can detect that (universal PRRSV-U is applicable to all pig blue-ear diseases poison to Pestivirus suis and pig blue-ear disease poison in sample to be measured simultaneously, high pathotype PRRSV-M is applicable to detect high-pathogenicity porcine reproductive and respiratory syndrome virus) two large class pathogenic agent, and accurately can detect in real time and carry out quantitatively to virus, it is very convenient to apply.Because the method introduces specificity amplification primer and fluorescent probe, the sensitivity of detection and specificity are strengthened significantly, thus avoid that other detection method specificitys are not high easily fails to pinpoint a disease in diagnosis the problem with mistaken diagnosis, based on above-mentioned advantage, this test kit is adapted at applying of the extensive examination such as animal health supervision at different levels institute, animal epidemic prevention and control central animals hospital and various Animal diseases research institutions.
The test kit obtained according to present method principle, can detect that Pestivirus suis and pig blue-ear disease poison and highly pathogenic PRRS are malicious easily, accuracy rate is high, is with a wide range of applications simultaneously; And easy and simple to handle, high specificity, has very strong practical value to the quick diagnosis of clinical sample and epidemiology survey.
Accompanying drawing explanation
Fig. 1 is the sensitivity experiments that in multicolor fluorescence PCR system, CSFV detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l;
Fig. 2 is the sensitivity experiments that in multicolor fluorescence PCR system, PRRSV-U detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l;
Fig. 3 is the sensitivity experiments that in multicolor fluorescence PCR system, PRRSV-M detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l;
Fig. 4 is the specificity experiments figure of CSFV in multicolor process PCR system, CSFV is positive, and PRRSV-CH1R strain, PRRSV-JXA1 strain, PRRSV-HuN4 strain, BVDV, SIV-H1N1, FMDV-O Mya98, JEV and negative quality control product are feminine gender;
Fig. 5 PRRSV-CH-1R strain is the positive, PRRSV-HuN4 strain is the positive, PRRSV-JXA1 strain is the positive, and BVDV, SIV-H1N1, FMDV-O Mya98, JEV and negative quality control product are feminine gender;
Fig. 6 PRRSV-HuN4 strain is the positive, PRRSV-JXA1 strain is the positive, and all the other samples and negative quality control product are feminine gender.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
embodiment 1
1, the design of Auele Specific Primer and probe
According to the Pestivirus suis retrieved from Genbank (Classical Swine Fever Virus, and the nucleotide sequence of PRRS virus (Reproductive and Respiratory Syndrome Virus) CSFV), be designed for the primer and probe that detect Pestivirus suis and pig blue-ear disease poison, its sequence is as follows:
Pestivirus suis (CSFV)
Upstream primer: CSFV-F:5 '-CAGTAGTTCGACGTGAGCAGAAG-3 ' (SEQ ID NO.1);
Downstream primer: CSFV-R:5 '-CGCTAGGGTTAAGGTGTGTCTTG-3 ' (SEQ ID NO.2);
Fluorescent probe: CSFV-P:5 '-ACCTCGAGATGCTACGTGGACGA-3 ' (SEQ ID NO.3), expanding fragment length 81bp;
PRRS virus is universal:
Upstream primer: PRRSV-UF:5 '-GGTTGGCTTTTGCTGTTGGT-3 ' (SEQ ID NO.4);
Downstream primer: PRRSV-UR:5 '-TCTGGCGAGTCAAACTCACAA-3 ' (SEQ ID NO.5);
Fluorescent probe: PRRSV-UP:5 '-VIC-TGTTCAAGCCTGTGTCCGACCCAG-BHQ1-3 ' (SEQ ID NO.6), expanding fragment length 76bp.
High-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M)
Upstream primer: PRRSV-MF:5 '-AGCTGATGACACCTTTGAGTGAGT-3 ' (SEQ ID NO.7);
Downstream primer: PRRSV-MR:5 '-GACAAATCCAGAGGCTCATCCT-3 ' (SEQ ID NO.8),
Fluorescent probe: PRRSV-MP:5 '-CY5-AGAACTGTGACAACAACGCTGACGCAC-BHQ2-3 ' (SEQ ID NO.9), expanding fragment length is 97bp.
Be FAM for detecting the fluorescent probe reporter group of Pestivirus suis, quenching group is BHQ1; Be VIC for detecting the fluorescent probe reporter group of PRRS virus (universal), quenching group is BHQ1; Be CY5 for detecting the fluorescent probe reporter group of high-pathogenicity porcine reproductive and respiratory syndrome virus, quenching group is BHQ2;
2, collection of specimens and pre-treatment
Present method and test kit are suitable for the tissue, serum, Secretory product etc. that specimen types comprises pig.Tissue sample: get under aseptic condition and organize about about 100 ~ 200mg, inserts in the clean EP pipe of 1.5ml, preserves to be checked.Serum: extract by inspection porcine vein 3-5ml with disposable sterilized injector, leave and take serum, preserve to be checked.Secretory product: gather under aseptic condition, preserves to be checked.The sample gathered should censorship as early as possible, or is stored in-20 DEG C.
3, the preparation of positive quality control product
Respectively with Pestivirus suis (CSFV), universal (PRRSV-U) RNA of PRRS virus and high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M) RNA standard substance for template, carry out pcr amplification, step is as follows:
Reverse transcription system:
Oligo dT Primer (50 μMs) 1 μ l, dNTP Mixture (10 mM) 1 μ l and total serum IgE 2 μ g, 65 DEG C of 5min, Quench, then adds 5 × PrimeScript Buffer 4 μ l(TAKARA company), RNase Inhibitor (40 U/ μ l) 0.5 μ l, PrimeScript RTase (200 U/ μ l) 1 μ l and RNase free H
2o 4.5 μ l
Reverse transcription condition:
30 DEG C of 10min, then 42 DEG C, 30min, carries out reverse transcription and becomes cDNA.
PCR system:
10 × multicolor fluorescence quantitative PCR Buffer 5 μ l, TaKaRa Taq(5 U/ μ l) each 2.5 mM of 1 μ l, dNTP Mixture() 4 μ l, each 0.5 μ l of above-mentioned CSFV, PRRSV-U and PRRSV-M upstream and downstream primer (10 μMs), above-mentioned cDNA0.5 μ l and RNase free H
2o39.25 μ l,
PCR condition:
According to 94 DEG C of 3 minutes denaturations, 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 45 seconds, totally 35 circulations, 72 DEG C extend 10 minutes.
Get after reaction 5 μ l pcr amplification products with 2% sepharose detect, the PCR primer of cutting glue purification is connected with pMD-18T carrier, recombinant plasmid pMD-18T-CSFV, pMD-18T-PRRSV-U and pMD-18T-PRRSV-M coat on culture dish, transformed competence colibacillus DH5 α cell, picking positive bacterium colony extracting plasmid, get product 5 μ l and dilute itself A260nm and A280nm absorbance of survey, calculate its concentration and be converted into absolute copy number, being diluted to 1.0 × 10
7copy/μ l, as the positive quality control product of multicolor fluorescence quantitative PCR.
Containing following composition: 500mMTris-HCl (pH8.0), 30mM MgCl in above-mentioned all 10 × multicolor fluorescence quantitative PCR buffer
2, 250mM KCl and 15% (v/v) the RNaseOut (U) of DMSO, 10U.
4, the extraction of viral RNA
1) solid tissue: get the glass homogenizer homogenate of about 0.5g tissue or scissors shreds, add 1.5ml physiological saline and continue grinding, go in the EP pipe of 1.5ml after homogenate, the centrifugal 2min of 10000rpm, get supernatant 100 μ l in the EP pipe of 1.5ml, add 200 μ l RNA extracting solution A fully to shake, leave standstill 3min;
2) serum or virus liquid: get 100 μ l serum and add 200 μ l RNA extracting solution A and fully shake, leaves standstill 3min, then adds 200 μ l RNA extracting solution B, firmly shake 15s, 4 DEG C of 13,000rpm centrifugal 5min;
3) abandon supernatant, add 1ml 75% ethanol, fully mix, the centrifugal 5min of 13,000rpm, carefully sucks most of residual liquid;
4) by uncovered for extraction tube in air at room temperature dry 10min, make to evaporate clean, precipitate by 20 μ l DEPC water dissolution and be viral RNA to be detected.
5, multicolor fluorescence quantitative pcr amplification
Each test reaction system is formulated as follows, and polychrome PCR MIX 20 μ l, ThermoScript II are 0.5 μ l, Taq enzyme is 0.5 μ l, brief centrifugation, then adds RNA 4 μ l to be detected; Equally the positive and negative control are set according to above-mentioned system, add positive quality control product or negative quality control product 4 μ l increases.
Wherein, the formula of PCR MIX is:
10 × multicolor fluorescence quantitative PCR buffer 2.5 μ l
CSFV-F（10μM） 1μl
CSFV-R（10μM） 1μl
PRRSV-UF（10μM） 1μl
PRRSV-UR（10μM） 1μl
PRRSV-MF（10μM） 1μl
PRRSV-MR（10μM） 1μl
CSFV-P（10μM） 0.5μl
PRRSV-UP（10μM） 0.5μl
PRRSV-MP（10μM） 0.5μl
dNTPS (10mM) 1μl
DdH
2o (not containing nuclease) 1.5 μ l
Total 20.0μl。
Each reaction tubes is put into the reactive tank of quantitative PCR instruments, the title and the fluorophor kind that arrange each detection (detect the reporter group selection FAM of CSFV, quenching group selection BHQ1; The reporter group detecting PRRSV-U selects VIC, and quenching group selects BHQ1; The reporter group detecting PRRSV-M selects CY5, quenching group selects BHQ2), setting cycling condition: the instrument cycling condition such as ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000, ABI PRISM7300/7500, MJ Opticon is 93 DEG C → 2 minutes, rear 93 DEG C 30 seconds → 55 DEG C 45 seconds, 40 circulations.LightCycler etc. use the instrument cycling condition of kapillary to be 93 DEG C → 2 minutes, rear 93 DEG C 5 seconds → 58 DEG C 45 seconds, totally 40 circulations.
6, interpretation of result and judgement
After reaction terminates, adjustment threshold value makes negative quality control product be 40 without Ct value or Ct value, FAM fluorescence Ct value be less than 35 circulations and S-type amplification curve for CSFV positive, VIC fluorescence Ct value be less than 35 circulations and S-type amplification curve for PRRSV-U positive, CY5 fluorescence Ct value be less than 35 circulations and S-type amplification curve be the PRRSV-M positive.Multiple channel fluorescence Ct value is less than 35 circulations and S-type amplification curve, be then that the test item of respective channel is common anode type.
When CY5 fluorescence Ct value is less than 35 circulations and S-type amplification curve, VIC fluorescence Ct value is more than or equal to 35 circulations or does not have S-type amplification curve, needs to recheck.
7, sensitivity experiment
Above-mentioned positive quality control product (10
7copy/μ l), dilution is 1.0 × 10 successively
6, 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3, 1.0 × 10
2, 1.0 × 10
1, 1.0 × 10
0copy/μ l, carries out sensitivity experiment.
The sensitivity experiments that in multicolor fluorescence quantitative PCR system, CSFV detects is shown in Fig. 1, and the susceptibility that in multicolor fluorescence quantitative PCR system, PRRSV-U detects is shown in Fig. 2, and the susceptibility that in multicolor fluorescence quantitative PCR system, PRRSV-M detects is shown in Fig. 3.Fig. 1 is the sensitivity experiments that in multicolor fluorescence PCR system, CSFV detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l.Fig. 2 is the sensitivity experiments that in multicolor fluorescence PCR system, PRRSV-U detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l.Fig. 3 is the sensitivity experiments that in multicolor fluorescence PCR system, PRRSV-M detects, and is from left to right followed successively by 1 × 10
8, 1 × 10
7, 1 × 10
6, 1 × 10
5, 1 × 10
4, 1 × 10
3, 1 × 10
2the amplification of the standard substance of copy/μ l.
Result proved test kit detection sensitivity is: 1.0 × 10
2μ l
-1, the method susceptibility of this multicolor fluorescence quantitative PCR is 200 times of regular-PCR, and accuracy is better than regular-PCR method.
8, specificity experiments
According to above-mentioned multicolor fluorescence quantitative PCR detecting method, with wild strains of classical swine fever virus (CSFV), PRRS virus american type vaccine strain (PRRSV-CH1R), high PRRS virus vaccine strain PRRSV-HuN4 and PRRSV-JXA1 that cause a disease, and other virus is as bovine viral diarrhoea (BVDV), porcine influenza H1N1 (SIV-H1N1), foot and mouth disease virus O type Burma 98 strain (FMDV-O Mya98), Latex agglutination test (JEV), negative control carries out confirmatory experiment and carries out sequence verification to product, result confirms, the inventive method and test kit specificity good, there is not false negative or false positive, the goodness of fit is 100%, experimental result is shown in Fig. 4, Fig. 5, Fig. 6
.
Fig. 4 is the specificity experiments figure of CSFV in multicolor process PCR system, as seen from the figure, CSFV is positive, and PRRSV-CH1R strain, PRRSV-JXA1 strain, PRRSV-HuN4 strain, BVDV, SIV-H1N1, FMDV-O Mya98, JEV and negative quality control product are feminine gender.
Fig. 5 is the specificity experiments of PRRSV-U in multicolor fluorescence PCR method, as seen from the figure, PRRSV-CH-1R strain is the positive, PRRSV-HuN4 strain is the positive, PRRSV-JXA1 strain is the positive, and BVDV, SIV-H1N1, FMDV-O Mya98, JEV and negative quality control product are feminine gender.
Fig. 6 is the specificity experiments of PRRSV-M in multicolor fluorescence PCR method, as seen from the figure, PRRSV-HuN4 strain is the positive, PRRSV-JXA1 strain is the positive, all the other samples and negative quality control product are feminine gender.
<110> Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120> mono-kind detects universal and three looks that highly pathogenic PRRS is malicious of Pestivirus suis, pig blue-ear disease poison simultaneously
Quantitative fluorescent PCR associated detecting method and test kit thereof
<130>
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
The artificial primer of <213>
<400> 1
cagtagttcg acgtgagcag aag 23
<210> 2
<211> 23
<212> DNA
The artificial primer of <213>
<400> 2
cgctagggtt aaggtgtgtc ttg 23
<210> 3
<211> 23
<212> DNA
The artificial primer of <213>
<400> 3
acctcgagat gctacgtgga cga 23
<210> 4
<211> 20
<212> DNA
The artificial primer of <213>
<400> 4
ggttggcttt tgctgttggt 20
<210> 5
<211> 21
<212> DNA
The artificial primer of <213>
<400> 5
tctggcgagt caaactcaca a 21
<210> 6
<211> 24
<212> DNA
The artificial primer of <213>
<400> 6
tgttcaagcc tgtgtccgac ccag 24
<210> 7
<211> 24
<212> DNA
The artificial primer of <213>
<400> 7
agctgatgac acctttgagt gagt 24
<210> 8
<211> 22
<212> DNA
The artificial primer of <213>
<400> 8
gacaaatcca gaggctcatc ct 22
<210> 9
<211> 27
<212> DNA
The artificial primer of <213>
<400> 9
agaactgtga caacaacgct gacgcac 27
Claims (10)
1. the universal and three fluorescence quantitative PCR associated detecting method that highly pathogenic PRRS is malicious of Pestivirus suis, pig blue-ear disease poison, is characterized in that: comprise the steps:
1) viral RNA in testing sample is extracted;
2) with the total serum IgE obtained for template, use Pestivirus suis (CSFV)
Upstream primer: CSFV-F:5 '-CAGTAGTTCGACGTGAGCAGAAG-3 ' (SEQ ID NO.1);
Downstream primer: CSFV-R:5 '-CGCTAGGGTTAAGGTGTGTCTTG-3 ' (SEQ ID NO.2);
Fluorescent probe: CSFV-P:5 '-ACCTCGAGATGCTACGTGGACGA-3 ' (SEQ ID NO.3)
Universal with PRRS virus:
Upstream primer: PRRSV-UF:5 '-GGTTGGCTTTTGCTGTTGGT-3 ' (SEQ ID NO.4);
Downstream primer: PRRSV-UR:5 '-TCTGGCGAGTCAAACTCACAA-3 ' (SEQ ID NO.5);
Fluorescent probe: PRRSV-UP:5 '-VIC-TGTTCAAGCCTGTGTCCGACCCAG-BHQ1-3 ' (SEQ ID NO.6);
With high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV-M)
Upstream primer: PRRSV-MF:5 '-AGCTGATGACACCTTTGAGTGAGT-3 ' (SEQ ID NO.7);
Downstream primer: PRRSV-MR:5 '-GACAAATCCAGAGGCTCATCCT-3 ' (SEQ ID NO.8),
Fluorescent probe: PRRSV-MP:5 '-CY5-AGAACTGTGACAACAACGCTGACGCAC-BHQ2-3 ' (SEQ ID NO.9);
Carry out three fluorescence quantitative PCR detection, wherein, Pestivirus suis, PRRS virus are universal, the fluorescent probe reporter group of high-pathogenicity porcine reproductive and respiratory syndrome virus is not identical;
3) according to the fluorescence Ct value of testing sample, interpretation of result: after reaction terminates, judges whether testing sample is that CSFV, PRRSV-U, PRRSV-M are positive.
2. associated detecting method according to claim 1, it is characterized in that: the reaction system cumulative volume of described quantitative fluorescent PCR reaction is 25 μ l, composed as follows: the swine fever that multicolor fluorescence quantitative PCR MIX 20 μ l, ThermoScript II are 0.5 μ l, Taq enzyme is 0.5 μ l and extraction and PRRS virus RNA or positive quality control product or negative quality control product 4 μ l; Wherein contain 10 × multicolor fluorescence quantitative PCR buffer, dNTPs in three fluorescence quantitative PCR MIX.
3. associated detecting method according to claim 2, is characterized in that: containing following composition: 500mMTris-HCl (pH8.0), 30mM MgCl in described 10 × multicolor fluorescence quantitative PCR buffer
2, 250mM KCl and 15% (v/v) the RNaseOut (U) of DMSO, 10U.
4. associated detecting method according to claim 2, is characterized in that: described multicolor fluorescence quantitative PCR MIX consists of:
10 × multicolor fluorescence quantitative PCR buffer 2.5 μ l
CSFV-F（10μM） 1μl
CSFV-R（10μM） 1μl
PRRSV-UF（10μM） 1μl
PRRSV-UR（10μM） 1μl
PRRSV-MF（10μM） 1μl
PRRSV-MR（10μM） 1μl
CSFV-P（10μM） 0.5μl
PRRSV-UP（10μM） 0.5μl
PRRSV-MP（10μM） 0.5μl
dNTPS (10mM) 1μl
ddH
2O 1.5μl
Total 20.0μl。
5. associated detecting method according to claim 1, is characterized in that: multicolor fluorescence quantitative PCR reaction conditions is: 93 DEG C 2 minutes, then 93 DEG C 30 seconds, 55 ~ 58 DEG C 45 seconds, gather fluorescent signal, 40 circulations.
6. one kind is detected Pestivirus suis simultaneously, the three fluorescence quantitative PCR combined detection kit that pig blue-ear disease poison is universal and highly pathogenic PRRS is malicious, comprise following composition: viral nucleic acid extracting solution A, viral nucleic acid extracting solution B, ThermoScript II system, Taq enzyme system, multicolor fluorescence quantitative PCR MIX, positive quality control product, negative quality control product, it is characterized in that, containing 10 × multicolor fluorescence quantitative PCR buffer in described multicolor fluorescence quantitative PCR MIX, dNTPS, and Pestivirus suis, PRRS virus is universal, the Auele Specific Primer of high-pathogenicity porcine reproductive and respiratory syndrome virus and polychromatic probe, sequence is respectively:
Pestivirus suis:
Upstream primer: CSFV-F:5 '-CAGTAGTTCGACGTGAGCAGAAG-3 ' (SEQ ID NO.1);
Downstream primer: CSFV-R:5 '-CGCTAGGGTTAAGGTGTGTCTTG-3 ' (SEQ ID NO.2);
Fluorescent probe: CSFV-P:5 ' 6-Fam-ACCTCGAGATGCTACGTGGACGA-BHQ1-3 ' (SEQ ID NO.3);
PRRS virus is universal:
Upstream primer: PRRSV-UF:5 '-GGTTGGCTTTTGCTGTTGGT-3 ' (SEQ ID NO.4);
Downstream primer: PRRSV-UR:5 '-TCTGGCGAGTCAAACTCACAA-3 ' (SEQ ID NO.5);
Fluorescent probe: PRRSV-UP:5 '-VIC-TGTTCAAGCCTGTGTCCGACCCAG-BHQ1-3 ' (SEQ ID NO.6);
High-pathogenicity porcine reproductive and respiratory syndrome virus
Upstream primer: PRRSV-MF:5 '-AGCTGATGACACCTTTGAGTGAGT-3 ' (SEQ ID NO.7);
Downstream primer: PRRSV-MR:5 '-GACAAATCCAGAGGCTCATCCT-3 ' (SEQ ID NO.8),
Fluorescent probe: PRRSV-MP:5 '-CY5-AGAACTGTGACAACAACGCTGACGCAC-BHQ2-3 ' (SEQ ID NO.9);
Wherein, the fluorescent probe reporter group of Pestivirus suis is FAM, and quenching group is BHQ1; The universal fluorescent probe reporter group of PRRS virus is VIC, and quenching group is BHQ1; The fluorescent probe reporter group of high-pathogenicity porcine reproductive and respiratory syndrome virus is CY5, and quenching group is BHQ2.
7. according to the detection kit described in claim 6, it is characterized in that: containing following composition: 500mMTris-HCl (pH8.0), 30mM MgCl in 10 × multicolor fluorescence quantitative PCR buffer
2, 250mM KCl and 15% (v/v) the RNaseOut (U) of DMSO, 10U.
8. according to the detection kit described in claim 6, it is characterized in that: described multicolor fluorescence quantitative PCR MIX's is composed as follows:
10 × multicolor fluorescence quantitative PCR buffer 2.5 μ l
CSFV-F（10μM） 1μl
CSFV-R（10μM） 1μl
PRRSV-UF（10μM） 1μl
PRRSV-UR（10μM） 1μl
PRRSV-MF（10μM） 1μl
PRRSV-MR（10μM） 1μl
CSFV-P（10μM） 0.5μl
PRRSV-UP（10μM） 0.5μl
PRRSV-MP（10μM） 0.5μl
dNTPS (10mM) 1μl
ddH
2O 1.5μl
Total 20.0μl。
9. according to the detection kit described in claim 6, it is characterized in that: contain in the every 1000ml of described viral extract A: guanidinium isothiocyanate 480g, 0.1M Tris-HCl(pH 6.4) 500ml, 0.2M EDTA(pH8.0) 120ml, surplus is water.
10. according to the detection kit described in claim 6, it is characterized in that: described viral extract B consists of Virahol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742262.5A CN104531897A (en) | 2014-12-05 | 2014-12-05 | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410742262.5A CN104531897A (en) | 2014-12-05 | 2014-12-05 | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104531897A true CN104531897A (en) | 2015-04-22 |
Family
ID=52847516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410742262.5A Pending CN104531897A (en) | 2014-12-05 | 2014-12-05 | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104531897A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319055A (en) * | 2016-08-30 | 2017-01-11 | 天津喜诺生物医药有限公司 | Triple nucleic acid test kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212623A (en) * | 2011-06-02 | 2011-10-12 | 广东省农业科学院兽医研究所 | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof |
| CN102373297A (en) * | 2011-08-04 | 2012-03-14 | 河南农业大学 | CSFV (Classical Swine Fever Virus), PRRSV (Porcine Reproductive and PCV2 (Porcine Circovirus 2) multiplex SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) primer and detection method thereof |
| CN102816865A (en) * | 2011-10-31 | 2012-12-12 | 武汉大学 | H1N1, PRRSV and CSFV quadruple detection kit |
-
2014
- 2014-12-05 CN CN201410742262.5A patent/CN104531897A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212623A (en) * | 2011-06-02 | 2011-10-12 | 广东省农业科学院兽医研究所 | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof |
| CN102373297A (en) * | 2011-08-04 | 2012-03-14 | 河南农业大学 | CSFV (Classical Swine Fever Virus), PRRSV (Porcine Reproductive and PCV2 (Porcine Circovirus 2) multiplex SYBR Green I real-time fluorescence PCR (Polymerase Chain Reaction) primer and detection method thereof |
| CN102816865A (en) * | 2011-10-31 | 2012-12-12 | 武汉大学 | H1N1, PRRSV and CSFV quadruple detection kit |
Non-Patent Citations (1)
| Title |
|---|
| 王晓波 等: "猪瘟病毒和猪繁殖与呼吸综合征病毒高致病性经典毒株多重RT-PCR方法的建立及应用", 《中国畜牧兽医学会2014年学术年会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319055A (en) * | 2016-08-30 | 2017-01-11 | 天津喜诺生物医药有限公司 | Triple nucleic acid test kit |
| CN106319055B (en) * | 2016-08-30 | 2019-12-13 | 天津喜诺生物医药有限公司 | triple nucleic acid detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Donin et al. | First report of Porcine teschovirus (PTV), Porcine sapelovirus (PSV) and Enterovirus G (EV-G) in pig herds of Brazil | |
| Deka et al. | Detection of bovine herpesvirus-1 infection in breeding bull semen by virus isolation and polymerase chain reaction | |
| CN102212623B (en) | Two-color fluorescence quantitative polymerase chain reaction (PCR) combined detection method of swine fever virus and blue ear disease virus and kit thereof | |
| CN104789697A (en) | Four-colored fluorescent quantitative PCR method capable of simultaneously detecting CSFV, PRRSV, PRV and PCV2 and reagent kit thereof | |
| CN108384899B (en) | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof | |
| CN106244725B (en) | Taqman real-time fluorescence PCR kit for diagnosing wild strain of porcine umbilical cord blood pseudorabies virus and application thereof | |
| Ciulli et al. | Evidence for Tunisian‐like pestiviruses presence in small ruminants in Italy since 2007 | |
| CN104745726B (en) | PRRS virus, high-pathogenicity porcine reproductive and respiratory syndrome virus and CSFV triple fluorescent quantitative detection kit | |
| CN103160615A (en) | Multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method | |
| CN106435033A (en) | Dual real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting and identifying wild strain and vaccine strain of CSFV (classical swine fever virus) in swine umbilical cord blood and application of dual real-time fluorescence RT-PCR kit | |
| Mahmoud et al. | Prevalence of bovine herpesvirus-1 in sheep and goats in Egypt | |
| CN110592278A (en) | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV | |
| CN106435032B (en) | Duplex RT-PCR primer, kit and method for simultaneously amplifying North American type and European type porcine reproductive and respiratory syndrome viruses | |
| CN112899404A (en) | Nested-RAA detection primer pair of mandarin frog iridovirus and application and detection method thereof | |
| CN103757137B (en) | Common primer nucleic acid amplification method for detecting three pig viruses synchronously and kit | |
| CN104531897A (en) | Method and kit for three-color fluorescent PCR detection of Classical swine feine virus (CSFV), porcine reproductive and respiratory syndrome virus-U (PRRSV-U) and porcine reproductive and respiratory syndrome virus-M (PRRSV-M) | |
| RU2607025C1 (en) | Synthetic oligonucleotide primers and method for detecting rna atypical pestivirus of cattle | |
| Hameed et al. | Screening the post-larvae of Macrobrachium rosenbergii for early detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by RT–PCR and immunological techniques | |
| CN106834539A (en) | Highly pathogenic PRRSV and CSFV simultaneous quantitative detection kit | |
| CN106435012A (en) | Real-time fluorescent PCR (polymerase chain reaction) kit for detecting swing Isospora in piglet umbilical cord blood and application thereof | |
| CN105779659A (en) | Primer and kit used for detecting foot-and-mouth disease virus type O as well as using method and application of kit | |
| CN105925727A (en) | Primer and kit for detecting foot and mouth disease virus type A and type O mixed infection and use method and application of kit | |
| Lipowski | Serological study on bovine viral diarrhoea virus infection in pig population in Poland between 2008 and 2011 | |
| CN110878379A (en) | Matched identification and detection method for attenuated live vaccine similar to NADC30 PRRSV and application thereof | |
| Thür et al. | Comparative detection of classical swine fever virus in striated muscles from experimentally infected pigs by reverse transcription polymerase chain reaction, cell culture isolation and immunohistochemistry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150422 |
|
| RJ01 | Rejection of invention patent application after publication |