CN110273024A - Fluorescence quantitative PCR detection primer and its kit based on Porcine epidemic diarrhea virus M gene - Google Patents
Fluorescence quantitative PCR detection primer and its kit based on Porcine epidemic diarrhea virus M gene Download PDFInfo
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- CN110273024A CN110273024A CN201910378503.5A CN201910378503A CN110273024A CN 110273024 A CN110273024 A CN 110273024A CN 201910378503 A CN201910378503 A CN 201910378503A CN 110273024 A CN110273024 A CN 110273024A
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- 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
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- 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
- C12Q1/701—Specific hybridization probes
Abstract
The invention discloses the fluorescence quantitative PCR detection primers based on Porcine epidemic diarrhea virus M gene, including upstream primer qPEDV-MF and downstream primer qPEDV-MR, they distinguish the base sequence of SEQ.ID.NO.1 and SEQ.ID.NO.2 in sequence table.Accordingly, inventor also develops the kit for facilitating diagnosis, and establishes corresponding detection method.Experimental study shows the checkout and diagnosis that the present invention is used for Porcine epidemic diarrhea virus, has preferable specificity, sensibility and repeatability, detection need to only can be completed in 3 hours, quick time saving, at low cost, the quick detection of all genotype of PEDV suitable for clinical sample.
Description
Technical field
The invention belongs to Porcine epidemic diarrhea virus to detect technical field of gene detection, more particularly to be based on pig epidemic
The fluorescence quantitative PCR detection primer and its kit of diarrhea virus M gene.
Background technique
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus
The acute infectious intestinal disease of a boar caused by (Porcine epidemic diarrhea virus, PEDV), clinical main table
It is now watery diarrhea and serious dehydration, it is the master for leading to piglet Deaths that the disease incidence of nursery-age pig, which is up to 80~100%,
Want one of epidemic disease.The disease is reported in Britain for 1976 for the first time, has broken out epidemic situation in Asian countries such as China, Thailand within 2010,
There is large-scale prevalence in the U.S. within 2013, causes huge economic loss to pig breeding industry.China divided for the first time in 1980
From PEDV is obtained, the generation for having the disease is reported in many areas in succession later.2016, in waiting people quietly to 17 province symptom of diarrhea
1272 parts of fecal samples of pig and asymptomatic pig have carried out PEDV positive detection, and diarrhea sample PEDV positive rate is 28.93%, this table
Bright PEDV is increasingly severe in the situation that China infects, therefore establishes a kind of time saving effective, easy to operate, at low cost method pair
PEDV is monitored and detects, and is of great significance.
PEDV is coronaviridae (Coronaviridae), and α coronavirus genus (Alpha coronavirus) belongs to sub-thread
Positive chain RNA virus has cyst membrane, and has the petal-shaped fibre to radially distribute prominent on cyst membrane.Porcine epidemic diarrhea virus particle
Shape has pleomorphism, and based on spherical shape, diameter range 95nm-190nm is about 130nm.Whole gene group size is about
28kb, structure are followed successively by 5 ' UTR-ORF1-S-ORF3-E-M-N-3 ' UTR, including four kinds of structural proteins from 5 ' ends to 3 ' ends, point
It Wei not spike protein (S), small membrane gene (E), albumen (M) and nucleocapsid protein (N) in film.Wherein, the overall length of M gene is
681bp encodes the membrane glycoprotein being made of 226 amino acid, is the key protein in virion assembling process.M albumen is made
For a membrane glycoprotein, the conservative of height ensure that the consistency of the completion that viral genetic is evolved and host's identification.It
And the combination of virus nucleocapsid is signal very crucial in Coronavirus particles assembling process, is risen to the assembly maturation of virus
Important function.
Summary of the invention
The technical problem to be solved in the present invention is to provide high specificity, high sensitivity, it is reproducible based on pig popularity
The fluorescence quantitative PCR detection primer and its kit of diarrhea virus M gene, all genotype of PEDV suitable for clinical sample
Quickly detection.
In order to solve the above technical problems, the invention adopts the following technical scheme:
Based on the fluorescence quantitative PCR detection primer of Porcine epidemic diarrhea virus M gene, including upstream primer qPEDV-MF and
Downstream primer qPEDV-MR, they are respectively provided with following base sequence:
Upstream primer qPEDV-MF:5 '-GGAATTTCACATGGAATATCA-3 '；
Downstream primer qPEDV-MR:5 '-CCATAGAATAGCCATCTTGAC-3 '.
The PCR detection primer of claim 1 is to for expanding the M gene in Porcine epidemic diarrhea virus.
Based on the fluorescent quantificationally PCR detecting kit of Porcine epidemic diarrhea virus M gene, contain upstream primer qPEDV-MF
With downstream primer qPEDV-MR, they are respectively provided with following base sequence:
Upstream primer qPEDV-MF:5 '-GGAATTTCACATGGAATATCA-3 '；
Downstream primer qPEDV-MR:5 '-CCATAGAATAGCCATCTTGAC-3 '.
Mentioned reagent box, including following reagent: positive control standard items, PCR reaction solution, negative control；Positive control mark
Quasi- product are the recombinant plasmid containing PEDV gene order, and PCR reaction solution includes upstream primer qPEDV-MF and downstream primer
qPEDV-MR、(2x)TB GreenTM Premix Ex TaqTMII, negative control are distilled water.
Recombinant plasmid is using pMD-18T as carrier.
The molar ratio of upstream primer qPEDV-MF and downstream primer qPEDV-MR are 1:1.
Content of the mentioned reagent box for quantitative fluorescent PCR measurement Porcine epidemic diarrhea virus M gene.
Current Porcine epidemic diarrhea virus prevention and treatment there are aiming at the problem that, inventor design and be prepared for based on pig popularity
The fluorescence quantitative PCR detection primer of diarrhea virus M gene, including upstream primer qPEDV-MF and downstream primer qPEDV-MR, it
Respectively in sequence table SEQ.ID.NO.1 and SEQ.ID.NO.2 base sequence.Accordingly, inventor, which also develops, facilitates diagnosis
Kit, and establish corresponding detection method.Experimental study shows the detection that the present invention is used for Porcine epidemic diarrhea virus
Diagnosis has preferable specificity, sensibility and repeatability, and detection need to only can be completed in 3 hours, quick time saving, cost
It is low, the quick detection of all genotype of PEDV suitable for clinical sample.
The PEDV fluorescence quantitative detecting method established accordingly, detection sensitivity 3.2*102Copy/μ L, Ct value and plasmid
Good linear relationship is presented between the logarithm of copy number, coefficient R 2=0.997 has repeatability well.Test knot
Fruit shows that the method detection sensitivity is 100 times higher than Standard PCR.Using different virus (porcine rotavirus, 9 type of pig enterovirus,
Porcine pseudorabies virus, porcine circovirus 2 type) DNA as template sets negative and positive control, fluorogenic quantitative detection is carried out,
Only have positive control amplification curve occur as the result is shown, other do not occur amplification curve, show the method high specificity.It is same anti-
System is answered to carry out batch interior repetition, each sample carries out 3 repetitions, and (fluorescence signal in i.e. each reaction tube reaches setting to Ct value
Thresholding when recurring number experienced) coefficient of variation is respectively 0.17%, 0.44%, 0.60%, respectively less than 1%, show error
All very small, amplification efficiency is stablized.
Detailed description of the invention
Fig. 1 is fluorescent quantitation amplification kinetic curve schematic diagram of the present invention.
Fig. 2 is fluorescent quantitation standard curve schematic diagram of the present invention.
Fig. 3 is the electrophoretogram of regular-PCR detection method, in figure: M:DL2000DNA Marker；1: positive；2: negative right
According to.
Fig. 4 is the electrophoretogram of regular-PCR detection sensitivity, in figure: M, DL2000DNA Marker；1,3.2*
107copies/μL；2,3.2*106copies/μL；3,3.2*105copies/μL；4,3.2*104copies/μL；5,3.2*
103copies/μL；6,320copies/μL；7,32copies/μL；8,3.2copies/μL；9,0.32copies/μL；10,
0.032copies/μL；11, negative control.
Fig. 5 is regular-PCR specific detection result figure, in figure: M, DL2000DNA Marker；1, negative control；2,
PCV2；3,PEDV；4,PRV；5,PEV-9；6,RV.
Fig. 6 is fluorescent quantitation specific detection result figure of the present invention, in figure: 1, negative control；2,PCV2；3,RV；4,
PRV；5,PEV-9；6,PEDV.
Specific embodiment
The foundation of embodiment 1, Porcine epidemic diarrhea virus fluorescence quantifying PCR method
(1) design and synthesis of primer
According to the M gene order of the GenBank Porcine epidemic diarrhea virus strain logged in, set using Primer5.0 software
1 pair of special primer is counted out, primer is synthesized by biological (Shanghai) Technology Co., Ltd. of outstanding Lee, specific as follows:
Upstream primer qPEDV-MF:5 '-GGAATTTCACATGGAATATCA-3 ' (sequence table SEQ .ID.NO.1)；
Downstream primer qPEDV-MR:5 '-CCATAGAATAGCCATCTTGAC-3 ' (sequence table SEQ .ID.NO.2).
Expanding target fragment size is 107bp (sequence table SEQ .ID.NO.3).
(2) clone of M gene
According to the PEDV positive sample that inventor saves, according to AxyPrep body fluid viral DNA/RNA small volume of reagent box, into
The extraction of row total serum IgE, reverse transcription, total cDNA sample are saved to -80 DEG C, and using the reaction system of 25 μ L, reaction condition is 94 DEG C
Initial denaturation 3min, then expanded with 40 circulations, every circulation is with 94 DEG C of denaturation 25s, and 55 DEG C of extension 25s, last 72 DEG C extend
8min.After the completion of amplification, 10 μ L products is taken to be identified with 1.5% agarose gel electrophoresis.Positive PCR product is accredited as to use
Omega Gel ExtractionKit (20) plastic recovery kit carries out purification and recovery, is cloned into pMD-18T carrier and is transformed into
DH5 ɑ competent cell, picking positive colony, and carry out sequencing identification.
(3) foundation of standard curve
Recombinant plasmid is extracted with plasmid extraction kit, measures OD260nm value with ultraviolet specrophotometer, standard plasmid is dense
Degree is converted into copy number/μ L, and diluted final concentration is in 3.2*109- 3.28102 copy numbers/μ L, fluorescent quantitation reaction system are overall
Product is 25 μ L, including (2x) TB GreenTMPremix Ex TaqTMEach 1 μ L of 12.5 μ L of II, 10 μm of upstream and downstream primer, DNA
2 μ L of template, 8.5 μ L of aqua sterilisa.Reaction condition is as follows: 95 DEG C of initial denaturation 30s；Expanded again with 39 circulations, it is every circulation with
95 DEG C of 5s denaturation, 60 DEG C of extension 30s；95 DEG C of extension 10s rise to 95 DEG C from 65 DEG C, and every 5s increases by 0.5 DEG C.And it is followed each
Fluorescence signal is acquired at the end of the elongating temperature of ring to be measured in real time, and finally carries out melting curve analysis pcr amplification product
Specificity.Each dilution gradient does 3 repeating hole controls, is averaged the calculating coefficient of variation.
As a result: within the scope of diluted linear concentration, template quantity is in linearly related, correlation between corresponding to corresponding Ct value
Coefficient is 0.997.Ct value is respectively 34.46,30.37,26.64,22.58,18.94,15.13,11.52,9.08, standard curve
Slope be -3.6914, intercept 37.701, linear equation: y=-3.6914x+37.701.Wherein y indicates Ct value, and x is indicated
Log (viral load), unit are copy number/μ L.
(4) sensitivity experiments
The minimum content that Standard PCR can be detected is 3.2*104Copy number/μ L, and the lowest detection of quantitative fluorescent PCR
Amount is 3.2*102Copy number/μ L.Therefore, the quantitative fluorescent PCR sensibility with higher that the present invention establishes, it is quicker than Standard PCR
Sense degree is 100 times high.
(5) specificity experiments
Conventional RT-PCR detection is carried out to PEDV, RV, PEV9, PRV, PCV2, it is ensured that it is carries out fluorescence after positive sample
Quantitative pcr amplification.The results show that only PEDV amplification is the positive, ct value is 30.26, other samples are without specific amplification, table
Bright this method has stronger specificity.
(6) repeated experiment
Choose 3.2*103, 3.2*106, 3.2*108The positive criteria plasmid of these three various concentrations carries out 3 repetitions respectively
Property test.Amplification such as table 1, the coefficient of variation of three samples are respectively less than 1%, illustrate the fluorescence quantitative RT-PCR detecting method
Repeatability with higher.
The repetitive test result of 1 quantitative fluorescent PCR of table
2 practical application one of embodiment
In January, 2019, for Guangxi large-scale pig farm, environmental sample is sampled, and it is fixed to carry out fluorescence using the present invention
PCR detection is measured, determines viral level.As a result such as table 2 (unit: copy number/mL)
2 Guangxi large-scale pig farm fluorescence quantitative PCR detection PEDV of table
Conclusion: in piggery enviroment sample, in delivery room manure pit contain higher viral level, it is proposed that pig farm to delivery room manure pit into
Row cleaning and disinfection.
3 practical application two of embodiment
The PEDV strain isolated to inventor, the 6th withholds the virus liquid of collection, carries out fluorescent quantitation using the present invention
The measurement of PCR determines its viral copy number.As a result such as table 3 (unit: copy number/mL)
3 PEDV virus liquid quantitative fluorescent PCR measurement result of table
Conclusion: according to the viral copy number of measurement, can carry out continuing to pass on for the strain of subsequently selected high copy number, into
And obtain the higher strain of titre.
Sequence table
<110>Guangxi University
<120>fluorescence quantitative PCR detection primer and its kit based on Porcine epidemic diarrhea virus M gene
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggaatttcac atggaatatc a 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccatagaata gccatcttga c 21
<210> 3
<211> 107
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggaatttcac atggaatatc atactgacga tactacttgt agtgcttcag tatggccatt 60
acaagtactc tgcgttcttg tatggtgtca agatggctat tctatgg 107
Claims (7)
1. the fluorescence quantitative PCR detection primer based on Porcine epidemic diarrhea virus M gene, it is characterised in that including upstream primer
QPEDV-MF and downstream primer qPEDV-MR, they are respectively provided with following base sequence:
Upstream primer qPEDV-MF:5 '-GGAATTTCACATGGAATATCA-3 '；
Downstream primer qPEDV-MR:5 '-CCATAGAATAGCCATCTTGAC-3 '.
2. PCR detection primer described in claim 1 is to for expanding the M gene in Porcine epidemic diarrhea virus.
3. a kind of fluorescent quantificationally PCR detecting kit based on Porcine epidemic diarrhea virus M gene, it is characterised in that contain upstream
Primer qPEDV-MF and downstream primer qPEDV-MR, they are respectively provided with following base sequence:
Upstream primer qPEDV-MF:5 '-GGAATTTCACATGGAATATCA-3 '；
Downstream primer qPEDV-MR:5 '-CCATAGAATAGCCATCTTGAC-3 '.
4. kit according to claim 3, it is characterised in that including following reagent: positive control standard items, PCR reaction
Liquid, negative control；The positive control standard items are the recombinant plasmid containing PEDV gene order, and PCR reaction solution includes upstream
Primer qPEDV-MF and downstream primer qPEDV-MR, (2x) TB GreenTMPremix Ex TaqTMII, negative control are double steam
Water.
5. kit according to claim 4, it is characterised in that: the recombinant plasmid is using pMD-18T as carrier.
6. kit according to claim 4, it is characterised in that: the upstream primer qPEDV-MF and downstream primer
The molar ratio of qPEDV-MR is 1:1.
7. content of the kit as claimed in claim 3 for quantitative fluorescent PCR measurement Porcine epidemic diarrhea virus M gene.
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Cited By (2)
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| CN112760418A (en) * | 2021-01-30 | 2021-05-07 | 福建傲农生物科技集团股份有限公司 | Primer and kit for porcine epidemic diarrhea virus and application of primer and kit |
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