CN103243179B - Shell-type PCR (polymerase chain reaction) amplification primer of porcine epidemic diarrhea virus and application thereof - Google Patents

Shell-type PCR (polymerase chain reaction) amplification primer of porcine epidemic diarrhea virus and application thereof Download PDF

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Publication number
CN103243179B
CN103243179B CN201310168452.6A CN201310168452A CN103243179B CN 103243179 B CN103243179 B CN 103243179B CN 201310168452 A CN201310168452 A CN 201310168452A CN 103243179 B CN103243179 B CN 103243179B
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epidemic diarrhea
diarrhea virus
porcine epidemic
pcr
shell
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CN103243179A (en
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王东东
宋延华
周庆丰
潘永飞
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Winson food group Limited by Share Ltd
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Guangdong Wens Foodstuff Group Co Ltd
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Abstract

The invention discloses a shell-type PCR (polymerase chain reaction) amplification primer of a porcine epidemic diarrhea virus and an application thereof, and belongs to the technical fields of animal virology and molecular biology. According to the shell-type PCR amplification primer of the porcine epidemic diarrhea virus disclosed by the invention, a nucleotide sequence is shown in SEQ ID NO:1-4. The primer can be used for preparing a kit for detecting the porcine epidemic diarrhea virus. Not only can classical PEDV (porcine epidemic diarrhea virus) be detected, but also a novel variant strain can be detected; the detection sensitivity is high; the shell-type PCR amplification primer can conveniently and rapidly detect samples such as clinical waste and intestinal contents; formulation of targeted prevention and control measures is facilitated; and the shell-type PCR amplification primer has a great effect on improvement of the PEDV prevention and control effect, and guarantee of stable pig-raising production.

Description

Sleeve type PCR amplimer and the application thereof of Porcine epidemic diarrhea virus
Technical field
The present invention relates to animal virology and technical field of molecular biology, be specifically related to sleeve type PCR amplimer and the application thereof of a kind of Porcine epidemic diarrhea virus.
Background technology
Porcine epizootic diarrhea (Porcine epidemic diarrhea, PED) be by Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) the acute contact infectious intestinal disease of one causing, principal character is diarrhoea, vomiting, dehydration and high mortality (Pensaert et al., A new coronavirus-like particle associated with diarrhea in swine. Arch Virol. 1978,58 (3): 243~247.; Ducatelle et al., Pathology of experimental CV777 coronavirus enteritis in piglets. I. Histological and histochemical study. Vet Pathol. 1982,19 (1): 46~56.).PEDV is a member of coronavirus genus, the not susceptible of weanling pig, and the lethality rate of this section of age in days swinery can reach 95%.
PEDV belongs to coronavirus genus I group, is RNA virus, and genome is sub-thread normal chain, non-segmented negative.Genome comprises 6 open reading frame (ORF), from 5 ' be followed successively by ORF1(20346 nt to 3 ' order); S gene (4152 nt); ORF3 gene (675 nt); E gene (231 nt); M gene (681 nt) and N gene (1326 nt) (Kocherhans et al.; Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence[J] .Virus Genes. 2001,23 (2): 137~144.; Brian et al., Coronavirus genome structure and replication. Curr Top Microbiol Immunol. 2005,287:1 ~ 30.).
Since 2010, a large-scale PEDV is popular in China's Mainland generation, has caused huge loss to pig industry.There is document to carry out careful analysis (Pan Y et al. to this popular PEDV strain and gene thereof; Isolation and characterization of a variant porcine epidemic diarrhea virus in China[J]. Virol J. 2012, Sep 12; 9:195.), found comparing S gene from classical strains in the early time and having some different gene expression characteristicses of variation strain.
Differentiate with classical Porcine epidemic diarrhea virus the quick method of distinguishing but also not having at present to make a variation.
Summary of the invention
The object of the invention is to for the problems referred to above of the prior art, by the analysis to Porcine epidemic diarrhea virus S gene, design two pairs of primers, and set up Nested Polymerase Chain Reaction, can differentiate efficiently differentiation to variation and classical Porcine epidemic diarrhea virus.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The sleeve type PCR amplimer that the invention provides Porcine epidemic diarrhea virus, nucleotide sequence is as follows:
F1:CACTTAGCCTACCACAAGATGTCA(SEQ ID NO:1),
R1:TCATTATCCCATGTTATGCCGA(SEQ ID NO:2);
F2:GGTGAAAACCAGGGTGTCAA(SEQ ID NO:3),
R2:TCGCGCAGTAGCATTAGTGTTA(SEQ ID NO:4)。
The Porcine epidemic diarrhea virus genome sequence logging according to NCBI, the accession number: (JN825712 of variation strain, JX088695, JX188454, JX489155, JX261936, JX112709, JX524137, JQ282909) and classical strains (AF353511, EF185992, Z25483, JQ023161, JN547228) information that provides, through compare of analysis, design the primer of above sleeve type PCR, used above-mentioned primer to carry out sleeve type PCR amplification, can differentiate classical strain and variant by electrophoresis, save the step of order-checking, saved time and cost.
Extract the RNA in detected sample, after reverse transcription, first use primers F 1 and R1 to carry out first round PCR, product stays a part to carry out agarose gel electrophoresis, another part continues as template, take turns PCR taking F2 and R2 as primer carries out second, amplified production is observed band after agarose gel electrophoresis, if first round PCR product only has the band of 458 bp, second takes turns and does not have, and shows to contain in sample to be checked classical PEDV; If first round PCR product only has the band of a 467bp, second takes turns the band that only has a 201bp, shows the PEDV that contains variation in sample to be checked; If two-wheeled PCR product does not all have band, show in sample to be checked not containing PEDV.
The present invention also provides the sleeve type PCR amplimer of above-mentioned Porcine epidemic diarrhea virus in the application of preparing in the reagent that detects classical Porcine epidemic diarrhea virus and/or Novel pig epidemic diarrhea virus.
The present invention also provides a kind of test kit that detects Porcine epidemic diarrhea virus, it is characterized in that, comprises following component:
(1) reverse transcription part:
RT premixed liquid 1: random primer and dNTP mix;
2:5 times of reverse transcription damping fluid of RT premixed liquid, RNA enzyme inhibitors, ThermoScript II, containing the distilled water of RNA enzyme;
(2) PCR part:
Overcoat PCR premixed liquid: archaeal dna polymerase, primers F 1 and R1, aseptic double-distilled water;
Inner sleeve PCR premixed liquid: archaeal dna polymerase, primers F 2 and R2, aseptic double-distilled water.
Compared with prior art, the present invention has following beneficial effect:
The invention provides the amplimer of a combined type PCR, this primer specificity and sensitivity are all higher, and can detect the PEDV of variation, can various clinical samples be detected, be differentiated, thereby the PEDV strain of pop is distinguished efficiently, simple to operate, practical.
Brief description of the drawings
Fig. 1: sample detection electrophorogram, swimming lane 1,2,3,4,5 is respectively the classical strain cell culture fluid of PEDV, PEDV variant cell culture fluid, enteron aisle pathological material of disease, ight soil pathological material of disease, DMEM.
Fig. 2: the PCR specificity inspection electrophorogram of distinguishing variation and classical Porcine epidemic diarrhea virus, A is first round pcr amplification product electrophoresis result, B second takes turns pcr amplification product electrophoresis result, swimming lane 1 is PEDV classical strains, swimming lane 2 is PEDV variation strain, and swimming lane 3-8 is respectively transmissible gastro-enteritis virus, porcine rotavirus, pig breeding and disordered breathing syndrome virus, pig circular ring virus, Pestivirus suis, PRV (Pseudorabies virus).
Fig. 3: the PCR method susceptibility inspection electrophorogram of distinguishing novel and classical Porcine epidemic diarrhea virus, shown in figure, be first round pcr amplification product electrophoresis result, wherein swimming lane 1,2,3,4,5,6,7 represents that respectively in sample to be checked, cDNA content is 1.58 μ g, 0.158 μ g, 0.0158 μ g, 1.58ng, 0.158ng, 0.0158ng, 1.58pg.
Embodiment
embodiment 1
(1) sample RNA to be checked extracts: 200 μ L cell culture fluids (infect autonomous isolated strain CHGD-01, this strain is through being accredited as the PEDV of variation), 200 μ L cell culture fluids (infect autonomous isolated strain ShQT, this strain is through being accredited as classical PEDV), enteron aisle pathological material of disease and 100mg ight soil (whether pathological material of disease and ight soil the unknown infect virus) and the DMEM(cell culture medium of 100mg, not containing PEDV) as sample to be checked, carry out respectively following operation:
Add 1mL PBS damping fluid fully to grind ,-20 DEG C of multigelations 3 times, centrifugal 10 min of 8000 rpm, get supernatant liquor 200 μ L, add 0.2mL chloroform, concussion mixes after 15s at room temperature (15 DEG C~30 DEG C) and places after 2~3min, 12000g(2 DEG C~8 DEG C) centrifugal 15min; Get upper strata water and be placed in new EP pipe, add 0.5mL Virahol, at room temperature (15 DEG C~30 DEG C) place 10min, 12000g(2 DEG C~8 DEG C) centrifugal 10min; Abandon supernatant, add 1mL 75% ethanol to wash, vortex mixed, 7500g(2 DEG C~8 DEG C) centrifugal 5min, abandons supernatant; Allow precipitation at room temperature seasoning of RNA after add 20 μ L Rnase-Free H 2o dissolves, and is RNA template.The RNA extracting is put in-80 DEG C of preservations.
(2) RNA extracting joins in reverse transcription premix reaction solution and carries out reverse transcription, obtains cDNA template.
Reverse transcription system and process: the RNA of 8 μ L and 2 μ L RT premixed liquids 1 mix, 65 DEG C of reaction 5min, are then placed in rapidly 2min on ice.This reaction mixture joins in RT premixed liquid 2 again, mixes and is placed on PCR instrument, by following conditioned response: 30 DEG C of 10min; 42 DEG C of 60min; 70 DEG C of 15min(are without circulation).
Wherein, RT premixed liquid 1:Ramdom 9 mer(50 μ M) and dNTP mix(10mM) each 1 μ L; RT premixed liquid 2:5 × PrimeScirpt Buffer 4 μ L, Rnase Inhibitor (40U/ μ L) 0.5 μ L, PrimeScript Reverse Transcriptase(200U/ μ L) 1 μ L, Rnase free H 2o 4.5 μ L, all purchased from Dalian TaKaRa company.
(3) 2 μ L cDNA templates are joined in overcoat PCR premixed liquid, preparation 25 μ L reaction systems, and mix.Wherein, overcoat PCR premixed liquid: Premix Taq(comprises DNA Polymerase1.25U/25 μ L; Buffer Tris-HCl, pH8.3 20mM, KCl 100mM, MgCl 23mM; The each 0.4mM of dNTP Mixture) 12.5 μ L, F1 and R1(are 20pmol) each 0.5 μ L, aseptic double-distilled water 9.5 μ L, all purchased from Dalian TaKaRa company.
(4) the PCR pipe of step (3) is placed in and on PCR instrument, carries out first round cyclic amplification reaction.Amplification condition is: 98 DEG C of denaturation 30s; Then 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1min carry out 35 circulations altogether; Last 72 DEG C are extended 10min.
(5) get 2 μ L first round PCR products as template, join in inner sleeve PCR premixed liquid, preparation 25 μ L reaction systems, mix.Inner sleeve PCR premixed liquid: Premix Taq(comprises DNA Polymerase1.25U/25 μ L; Buffer Tris-HCl, pH8.3 20mM, KCl 100mM, MgCl 23mM; The each 0.4mM of dNTP Mixture) 12.5 μ L, F2 and R2(are 20pmol) each 0.5 μ L, aseptic double-distilled water 9.5 μ L, all purchased from Dalian TaKaRa company.
Amplification condition is: 98 DEG C of denaturation 30s; Then 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1min carry out 35 circulations altogether; Last 72 DEG C are extended 10min.
(6) two-wheeled PCR respectively gets 5 μ L reaction product at 1%(mass ratio) sepharose on carry out electrophoresis qualification.
(7) result is judged: the classical strain of first round PCR product size 458bp() or 467bp(variant), second takes turns PCR product size 201bp.What two-wheeled PCR product electrophoresis had that two product 467 bp and 201bp band occur simultaneously is variant epidemic diarrhea virus, and what have that 458 bp bands occur is classical Porcine epidemic diarrhea virus, and what occur without band is negative.Detected result is shown in Fig. 1 and table 1.
Table 1 sample detection result to be checked
Sample to be checked Electrophoretic band Amplification Sequence Identification
Cell culture fluid (infecting strain CHGD-01) 467bp and 201bp Variant
Cell culture fluid (infecting strain ShQT) 458bp Classical strain
Enteron aisle pathological material of disease 467bp and 201bp Variant
Ight soil 467bp and 201bp Variant
DMEM cell culture medium Without amplified band Nothing
embodiment 2 specificity inspections
to exist the pathological material of disease of classical strain (strain ShQT), variant (strain CHGD-01) of epidemic diarrhea virus as positive control, transmissible gastro-enteritis virus, porcine rotavirus, pig breeding are carried out specific detection with the nutrient solution of disordered breathing syndrome virus, pig circular ring virus, Pestivirus suis, PRV (Pseudorabies virus), use method and the primer of embodiment 1, result all fails to expand any band except positive control, positive pathological material of disease expands respectively two bands and a band through two-wheeled PCR, sees Fig. 2.Its sequence is respectively SEQ ID NO:5(variant first round pcr amplification product), the classical strain first round pcr amplification product of SEQ ID NO:6() and SEQ ID NO:7(variant second take turns pcr amplification product).
embodiment 3 sensitivity tests
Extract with classical strain in embodiment 1 (strain ShQT) the cDNA sterilizing distilled water obtaining and do the gradient dilution of 10 times, cDNA content is respectively 1.58 μ g, 0.158 μ g, 0.0158 μ g, 1.58ng, 0.158ng, 0.0158ng, 1.58pg as template, each extent of dilution is respectively got 2 μ L as template, detect by embodiment 1 method, observe positive band, calculate its susceptibility with the high dilution that occurs the template used amount of positive expection band, result shows that minimum detectable activity is 0.16 ng, sees Fig. 3.
SEQUENCE LISTING
<110> Guangdong Wen’S Foodstuffs Group Co., Ltd.
Sleeve type PCR amplimer and the application thereof of <120> Porcine epidemic diarrhea virus
<130>
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> artificial sequence
<400> 1
cacttagcct accacaagat gtca 24
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
tcattatccc atgttatgcc ga 22
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
ggtgaaaacc agggtgtcaa 20
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
tcgcgcagta gcattagtgt ta 22
<210> 5
<211> 467
<212> DNA
<213> artificial sequence
<400> 5
cacttagcct accacaagat gtcaccaggt gctcagctaa cactaatttt aggcggttct 60
tttcaaaatt taatgttcag gcgcctgcag ttgttgtact gggcggttat ctacctattg 120
gtgaaaacca gggtgtcaat tcaacttggt actgtgctgg ccaacatcca actgctagtg 180
gcgttcatgg tatctttctt agccatatta gaggtggtca tggctttgag attggcattt 240
cgcaagagcc ttttgaccct agtggttacc agctttattt acataaggct actaacggta 300
acactaatgc tactgcgcga ctgcgcattt gccagtttcc cagcattaaa acattgggcc 360
ccactgctaa taatgatgtt acaacaggtc gtaactgcct atttaacaaa gccatcccag 420
ctcatatgag tgaacatagt gttgtcggca taacatggga taatgat 467
<210> 6
<211> 458
<212> DNA
<213> artificial sequence
<400> 6
cactcagcct accacaagat gtcactaggt gccagtctac tactaacttt aggcggttct 60
tttcaaaatt taatgttcag gcacctgccg tcgtcgtttt gggtggttac ctacctagta 120
tgaactcttc tagctggtac tgtggcacag gcattgaaac tgctagtggc gttcatggta 180
tttttctcag ctacatcgat tctggtcagg gctttgagat tggcatttcg caagagccgt 240
ttgatcctag tggttaccag ctttatttac ataaggccac taatggtaac actaatgcta 300
ttgcacgact gcgcatttgc cagtttcccg ataataaaac attgggccct actgttaatg 360
atgttacaac aggtcgtaac tgcctattca acaaagccat tccagcttat atgcgtgatg 420
gaaaagatat tgttgtcggc ataacatggg ataatgat 458
<210> 7
<211> 201
<212> DNA
<213> artificial sequence
<400> 7
ggtgaaaacc agggtgtcaa ttcaacttgg tactgtgctg gccaacatcc aactgctagt 60
ggcgttcatg gtatctttct tagccatatt agaggtggtc atggctttga gattggcatt 120
tcgcaagagc cttttgaccc tagtggttac cagctttatt tacataaggc tactaacggt 180
aacactaatg ctactgcgcg a 201

Claims (3)

1. the sleeve type PCR amplimer of Porcine epidemic diarrhea virus, is characterized in that, nucleotide sequence is as follows:
F1:CACTTAGCCTACCACAAGATGTCA,
R1:TCATTATCCCATGTTATGCCGA;
F2:GGTGAAAACCAGGGTGTCAA,
R2:TCGCGCAGTAGCATTAGTGTTA。
2. the application of the sleeve type PCR amplimer of Porcine epidemic diarrhea virus in the reagent of the preparation classical Porcine epidemic diarrhea virus of detection and/or variation Porcine epidemic diarrhea virus described in claim 1.
3. a test kit that detects Porcine epidemic diarrhea virus, is characterized in that, comprises following component:
(1) reverse transcription part:
RT premixed liquid 1: random primer and dNTP mix;
2:5 times of reverse transcription damping fluid of RT premixed liquid, RNA enzyme inhibitors, ThermoScript II, containing the distilled water of RNA enzyme;
(2) PCR part:
Overcoat PCR premixed liquid: archaeal dna polymerase, primers F 1 claimed in claim 1 and R1, aseptic double-distilled water;
Inner sleeve PCR premixed liquid: archaeal dna polymerase, primers F 2 claimed in claim 1 and R2, aseptic double-distilled water.
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CN103667531A (en) * 2013-12-06 2014-03-26 湖北省农业科学院畜牧兽医研究所 FQ-PCR detection method for swine epidemic diarrhea virus (PEDV) and primer pair used therein
CN104531902B (en) * 2014-12-31 2017-05-31 洛阳普莱柯万泰生物技术有限公司 A kind of kit and preparation method thereof
CN105821159A (en) * 2016-04-20 2016-08-03 华南农业大学 Nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV
CN109439799A (en) * 2018-11-06 2019-03-08 江西农业大学 It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus
CN109735662A (en) * 2019-03-11 2019-05-10 吉林正业生物制品股份有限公司 Identify the primer sets and detection kit of Porcine epidemic diarrhea virus classics and variation strain
CN109825649A (en) * 2019-04-09 2019-05-31 广西大学 Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit
CN111154917B (en) * 2020-02-22 2022-12-27 南京农业大学 PCR primer and method for identifying porcine pseudorabies virus variant strain

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