CN105886667A - Detection kit for porcine epidemic diarrhea virus and detection method thereof - Google Patents
Detection kit for porcine epidemic diarrhea virus and detection method thereof Download PDFInfo
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- CN105886667A CN105886667A CN201610325218.3A CN201610325218A CN105886667A CN 105886667 A CN105886667 A CN 105886667A CN 201610325218 A CN201610325218 A CN 201610325218A CN 105886667 A CN105886667 A CN 105886667A
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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
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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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The invention belongs to the technical field of in-vitro molecular diagnosis of biological medicine, and particularly relates to a detection kit for porcine epidemic diarrhea virus based on strand displacement PCR technology and a detection method thereof. The kit comprises a linear reporter gene with a specific probe, a pair of upstream and downstream reverse primers, an enzyme liquid, a positive control and a negative control. Massive clinical tests indicate that the detection kit for porcine epidemic diarrhea virus (PEDV) can be used for effectively detecting PEDV, has an amplified band of about 250bp and a detection sensitivity of 1.0pg which are higher than those of conventional RT-PCR, does not have cross reaction with porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV) and other five porcine derived pathogens, has the advantages of single detection background, high sensitivity, strong specificity, good stability and the like, can be used for detecting the porcine epidemic diarrhea virus in excrement, intestines and content of pigs, and has a wide application prospect.
Description
Technical field
The invention belongs to biological medicine external molecular diagnostic techniques field, particularly relate to a kind of based on strand displacement round pcr
The detection kit of Porcine epidemic diarrhea virus and detection method.
Background technology
Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) is that single-stranded positive RNA is sick
Poison, belongs to I class coronavirus, because it can infect different sexes, age and the pig of kind by contact, causes with under acute water sample
Dysentery, vomit, the porcine epizootic diarrhea (porcine epidemic diarrhea, PED) of the pathogenetic feature such as dehydration, become current
One of main infection pathogen that harm world pig industry produces.
In China, since reported first PED in 1976, PEDV expands, especially year by year in China outbreak of epidemic region
Between 2010-2011 years, PEDV is wantonly plunderred on the pig farm in more than 10 provinces of south China, causes millions of infected pigs's death, in
Revealing the epidemic status of high incidence, high mortality, cause heavy blow to China's pig industry, how prevention and control PEDV becomes me
The major tasks of state scholar research.For a long time, due to PEDV, to cell cultivation, adaptation ability is weak, Reproduction Conditions is complicated, cell
Pathological changes produces the reasons such as inconspicuous, and the separation and Culture difficulty causing PEDV is big, limits this viral biology characteristic and disease
Carrying out of the research work such as prevention and control, until Sweden scholar Hoffmann M in 1988 et al. is by adding in passage culture fluid
Entering pancreatin, on Vero cell, continuous blind passage is successfully obtained the cell cultivation of PEDV.Subsequently, Japan, Korea S and China etc. are in succession
The separation and Culture of PEDV and the relevant report of the cultural character on Vero cell thereof occur, for in-vitro multiplication and the vaccine of PEDV
Development is laid a good foundation.Set up the premise that stable PEDV detection method is effective prevention and control viral disease, at present, for
The detection method of PEDV is a lot, but all there is shortcomings, and such as traditional virus purification, virus multiplication difficulty, CPE is inconspicuous,
And the test period is long, operation is wasted time and energy, and is not suitable for clinical quickly detection；And immunofluorescence, ELISA, neutralization test etc. are exempted from
Although the epidemic disease detection technique detection time is short, easy to operate, but causes immune detection owing to PEDV antigen type is many and variation fast
Sensitivity and specificity all ratios are relatively low, are unfavorable for disease surveillance and treatment in time；And conventional RT-PCR, multiplex PCR and quantitative PCR
Although there is sensitivity, advantage special, quick etc. detection technique, owing to PCR detection is easily polluted and High sensitivity, cause false positive
Higher with false negative rate；Quantitative PCR achieves closed tube analysis, effectively prevent the recontamination of amplification, but still there is specimen, examination
Pollution problem when agent and operating process.For the problems referred to above, the present invention provides a kind of pig based on strand displacement round pcr popular
Property diarrhea virus (PEDV) detection kit, for science bridle PEDV provide effective detection means.
Summary of the invention
A kind of based on strand displacement round pcr the pig that it is an object of the invention to overcome deficiency of the prior art and invent
The detection kit of epidemic diarrhea virus and detection method thereof.
The present invention is achieved in that the detection kit of a kind of Porcine epidemic diarrhea virus, it is characterised in that: this reagent
Box includes 1 wire, reporter gene with specific probe, and described reporter sequences is as shown in SEQ NO:1.
Further, this test kit also includes 1 pair of upstream and downstream reverse primer, reverse primer sequences such as SEQ NO:4 and SEQ
Shown in NO:5.
Further, described reporter gene and reverse primer are configured to PCR and detect reactant liquor, and test kit also includes enzyme
Liquid, positive control, negative control.
Described enzyme liquid is by AMV Reverse Transcriptase (Promega) 0.8 μ L, Tfl DNA
Polymerase (Promega) 0.6 μ L, Taq DNA Ligase(NEB) 0.6 μ L composition；
Reactant liquor, by 5 × Reaction Buffe 5 μ L, 10 × Taq DNA Ligase Reaction Buffer 2.5 μ
The random primer 6 aggressiveness 2.0 μ L of l, 25mM, reporter gene 1.0 μ L, 10mM dNTPs 2.5 μ L, 25mM MgSO4 1μL、10μ
M upstream reverse primer 1.0 μ L, 10 μMs of reverse downstream primer 1.0 μ L, H2O 4.0 μ L forms；
Described positive control C is Porcine epidemic diarrhea virus cell culture；
Described negative control D is sterilizing distilled water.
A kind of detection method of the detection kit of Porcine epidemic diarrhea virus, it is characterised in that: comprise the steps:
Step 1) extracts the nucleic acid of sample to be checked；
Step 2) detection of PEDV mono-step:
Prepare 25 μ L reaction systems: the nucleic acid 3 μ L of preparation, reactant liquor 20 μ L, enzyme liquid 2 μ L, do negative and positive comparison, mixing simultaneously
Reactant liquor, of short duration centrifugal collection liquid is at the bottom of pipe；Described testing conditions: carry out 70 DEG C of 7min on PCR cycle instrument successively, 42
DEG C 30min, 95 DEG C of 3 min, 1 circulation；95 DEG C of 50sec, 60 DEG C of 10 min, 5 circulations；95 DEG C of 50s, 53 DEG C of 50s,
72 DEG C of 50s carry out 25 circulations；
Step 3) takes step 2) product 8 μ L carry out electrophoresis detection with the agarose gel of 2%, observe testing result.
The invention have the advantages that
The present invention is directed to Standard PCR and the shortcoming of multiplex PCR detection, fully analyze PEDV Chinese epidemic strain M gene order
Conservative, according to the genome sequence (KP890336.1) of Henan variant CH/HNYF/14 in GenBank data base, designs 2
To specific probe, use Standard PCR technology to be marked on arabidopsis TOC1 gene two ends respectively, form wire, two ends with spy
The reporter gene of specific probes；During detection, the probe at reporter gene two ends and genes of interest to be checked through hybridization, breach filling-in and
Ring-type reporter gene is formed, it is achieved the displacement of chain after connection；Using 1 to reverse primer the most again, it is right that amplification reporter gene realizes
The indirect detection of genes of interest.Because reporter gene is big with gene genetic background difference to be checked, can effectively overcome Standard PCR detection easily
The problem polluted.
Shown by substantial amounts of clinical trial, use Porcine epidemic diarrhea virus (PEDV) the detection examination that above-mentioned technology manufactures
Agent box can effectively detect PEDV, and amplified band size is about 250bp, and detection sensitivity is up to 1.0pg, higher than conventional RT-PCR,
And with other 5 boar sources such as transmissible gastro-enteritis virus (TGEV), porcine rotavirus (porcine rotavirus, PoRV)
The former no cross reaction of sexually transmitted disease (STD), has detection background single, the advantage such as highly sensitive, high specificity, good stability, can be to diarrhoea pig
Feces, intestinal and content carry out Porcine epidemic diarrhea virus detection, and application prospect is wide.
Accompanying drawing explanation
Fig. 1 be reporter gene through amplified production through agarose gel electrophoresis detection figure.
Fig. 2 is the agarose gel electrophoresis detection figure that Porcine epidemic diarrhea virus (PEDV) detects.
Fig. 3 is that in embodiment 2, the agarose of the detection kit specific test of Porcine epidemic diarrhea virus (PEDV) coagulates
Gel electrophoresis detection figure.
Fig. 4 is that the agarose of the detection kit susceptiveness test of Porcine epidemic diarrhea virus (PEDV) in embodiment 3 coagulates
Gel electrophoresis detection figure.
Detailed description of the invention
Primer used in following example is by the synthesis of Dalian treasured biological (Takara) company, AMV Reverse
Transcriptase and Tfl DNA Polymerase is purchased from Promega company, and Taq DNA Ligase is purchased from NEB company,
Random primer, dNTPs and DL-2000 are purchased from Takara company.Unreceipted specific experiment condition in the following example, generally according to
Normal condition or according to the condition proposed by manufacturer.
Embodiment 1
The detection of Porcine epidemic diarrhea virus (PEDV)
1.1 labeled primers and the design of reverse primer
Labeled primer is mainly made up of two parts, and 3 ' ends of primer are complementary with reporter gene, for preparing linear probe labelling
Reporter gene；5 ' ends of primer are then specific probe sequence, are used for and gene recombination to be checked.The present invention chooses one section with to be checked
Gene is unrelated, or the arabidopsis TOC1 gene order that germline is farther out is as reporter gene, length about 200-500bp, take on it,
The part that sequence is labeled primer of each 18bp in two ends, downstream；Choose gene PEDV Henan variant CH/HNYF/14's to be checked
One section of conserved region of M gene order (length about 50-200bp), respectively designs a left side (L probe) and right (R probe) 2 probes, makees
For another part of labeled primer, L, R probe is added on respectively 5 ' ends of upstream and downstream labeled primer, collectively forms complete mark
Note primer, primer sequence is as shown in SEQ NO:2 and SEQ NO:3, and wherein, in primer, left side 14bp is for PEDV M gene
Probe sequence.
Reverse primer is mainly used in the ring-type reporter gene of Inverse PCR amplification, and this reverse primer is retrieved from reporter gene except visiting
Any two segments outside pin sequence, the general two segment sequences selecting reporter gene middle, the sense strand conduct of one section of the right
Inverse PCR forward primer, one section of left side antisense strand as inverse PCR downstream primer, primer sequence such as SEQ NO:4 and SEQ
Shown in NO:5.
The design of primer and probe has been designed by the primer-design software (Prime Premier5.0) of specialty.Primer
Synthesis completed by biological (Takara) company of Dalian treasured.
1.2 the preparation of reporter gene
Reporter gene is mainly obtained by standard PCR amplification, utilizes above-mentioned labeled primer, uses standard PCR amplification reporter gene.
PCR amplification template is recombiant plasmid pMD18-T-TOC1, and PCR reaction system: rTaq premixes enzyme 50 μ l, upstream and downstream primer (10 μ
M) each 4 μ l, plasmid template 1 μ L, add water and supply 100 μ l；PCR cycle condition: 95 DEG C of degeneration 30s, 50 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 30s, 30 circulations, obtain reporter gene, the sequence of described reporter gene such as SEQ NO:1.
As it is shown in figure 1, amplified production is after agarose gel electrophoresis detection confirmation is errorless, carries out amplified production and reclaim purification, adopt
Reclaiming test kit with TIANGEN Biotech's agarose gel, operating procedure is shown in description, adjusts report base
The concentration 10 μ g/mL of cause.
1.3 detection kit
Detection kit comprises enzyme liquid A, reactant liquor B, positive control C, negative control D and five kinds of components of DEPC solution, concrete group
Become and preservation condition be as shown in the table:
Table 1 test kit composition and preservation condition
| Numbering | Test kit forms | Volume | Preservation condition |
| 1 | Enzyme liquid A | 21μL | -20℃ |
| 2 | Reactant liquor B | 210μL | -20℃ |
| 3 | Positive control C | 1.2mL | -20℃ |
| 4 | Negative control D | 40μL | -20℃ |
| 5 | DEPC water E | 1000μL | -20℃ |
Wherein, enzyme liquid A is by AMV Reverse Transcriptase (Promega) 0.8 μ L；Tfl DNA Polymerase
(Promega) 0.6 μ L；Taq DNA Ligase(NEB) 0.6 μ L composition；
Reactant liquor B is by 5 × Reaction Buffe 5 μ L；10 × Taq DNA Ligase Reaction Buffer 2.5μ
l；The random primer 6 aggressiveness 2.0 μ L of 25mM；The reporter gene 1.0 μ L of M probe labelling；10mM dNTPs 2.5μL；25mM
MgSO4 1μL；10 μMs of upstream reverse primer 1.0 μ L；10 μMs of reverse downstream primer 1.0 μ L；H2O 4.0 μ L forms；Positive control
C is Porcine epidemic diarrhea virus cell culture；
Negative control D is sterilizing distilled water.
The detection of 1.4 Porcine epidemic diarrhea virus (PEDV)
The using method of Porcine epidemic diarrhea virus (PEDV) detection kit mainly comprises the steps:
Step 1) uses Trizol reagent method to extract the nucleic acid of sample to be checked: take the testing sample of 200 μ L, positive control C respectively
With negative control D in 1.5 μ L centrifuge tubes, add Trizol reagent 800 μ L, add 200 μ L chloroforms after mixing, acutely shake
Mixing；13000rpm is centrifuged 10 minutes, takes supernatant in 1.5 new μ L centrifuge tubes, adds isopyknic isopropanol, mixes rear chamber
Temperature is placed 10 minutes；13000rpm is centrifuged 10 minutes, adds 700 μ L 75% ethanol after removing supernatant, overturns and washs for several times；
13000rpm is centrifuged 5 minutes, dries after removing supernatant as far as possible, adds 20 μ L DEPC water E and dissolves nucleic acid.
Step 2) take the nucleic acid of testing sample, positive control and negative control that 3 μ L are prepared as stated above respectively, add
20 μ L reactant liquor B, add 2 μ L enzyme liquid A, make total reaction volume reach 25 μ L, mix reactant liquor, and of short duration centrifugal collection liquid is to pipe
The end；
Step 3) is by step 2) reactant liquor prepared is placed on PCR cycle instrument, carries out successively: 70 DEG C of 7min, 42 DEG C of 30min,
95 DEG C of 3 min, 1 circulation；95 DEG C of 50sec, 60 DEG C of 10 min, 5 circulations；95 DEG C of 50s, 53 DEG C of 50s, 72 DEG C of 50s
Carry out 25 circulations.
Step 4) takes the product 8 μ L of step 3) and carries out electrophoresis detection with the agarose gel of 2%, result such as Fig. 2 institute
Show.
5) result interpretation
Positive control has clear band near 250bp, and negative control does not has；
If measuring samples occurs clear band near 250bp, then it is judged as that PEDV infects；
If measuring samples without amplified band, is then judged as that PRRSV is negative near 250bp.
Embodiment 2
The detection kit specific test of Porcine epidemic diarrhea virus (PEDV)
According to the process of embodiment 1, respectively to Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV),
Porcine rotavirus (PoRV), swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and PRV (Pseudorabies virus)
(PRV) carry out PCR amplification, observe cross reaction situation, assess the specificity of this detection kit according to this.As it is shown on figure 3, agar
Sugar detected through gel electrophoresis result shows, only Porcine epidemic diarrhea virus (PEDV) sample has a specific amplification band, and and its
Its cause of disease no cross reaction, specificity is good, shows that the present invention is only capable of detecting Porcine epidemic diarrhea virus (PEDV), will not detect
Go out other pig borne virus, can be used to carry out Porcine epidemic diarrhea virus (PEDV) and infect detection.
Embodiment 3
The detection kit susceptiveness test of Porcine epidemic diarrhea virus (PEDV)
Prepared Porcine epidemic diarrhea virus (PEDV) total serum IgE is measured by ultramicrospectrophotometer, adjusts pig popular
Property diarrhea virus total rna concentration is the nucleic acid solution of 100 ng/ μ L, uses RNase-free H2O carries out 10 times to nucleic acid solution
Doubling dilution, formed 10-1-10-7Totally 7 gradients, take the diluent 3 μ L of each gradient, then according to the process of embodiment 1
Carry out PCR amplification, simultaneously with RNase-free H2O, as negative control, carries out sensitivity tests, and agarose gel electrophoresis is examined
Survey amplification, assess the susceptiveness of this detection kit according to this.
As shown in Figure 4, it is diluted to 10 when the nucleic acid solution of Porcine epidemic diarrhea virus (PEDV)-5, during content as little as 1pg,
Test kit remains to detect positive band, shows test kit that the present invention the provides minimum to Porcine epidemic diarrhea virus (PEDV)
Detection limit is the viral RNA 1pg.
The present invention is directed to Standard PCR and the shortcoming of multiplex PCR detection, fully analyze PEDV Chinese epidemic strain M gene sequence
The conservative of row, according to the genome sequence (KP890336.1) of Henan variant CH/HNYF/14 in GenBank data base, if
Meter 2 to specific probe, uses Standard PCR technology to be marked on arabidopsis TOC1 gene two ends respectively, formed wire, two ends with
The reporter gene of specific probe；During detection, the probe at reporter gene two ends and genes of interest to be checked are through hybridization, breach filling-in
Ring-type reporter gene is formed, it is achieved the displacement of chain after connecting；Use 1 reverse primer, amplification reporter gene are realized the most again
Indirect detection to genes of interest.Because reporter gene is big with gene genetic background difference to be checked, Standard PCR can be effectively overcome to detect
The problem easily polluted.
Sequence table
<110>Henan animal husbandry institute of economics
<120>detection kit of Porcine epidemic diarrhea virus and detection method thereof
<160> 5
<170> PatentIn Version 3.3
<210> 5
<211> 408
<212> DNA
<213>artificial sequence, intermediate sequence is arabidopsis TOC1 genetic fragment, and each 14bp in both sides is the spy for PEDV M gene
Pin sequence
<400> 1
CATAAGAGTG ATGCGGCGAC TTTGTAGGGA CACTGTACCG AAAAACCCTA GTTCTGATTT 60
GGCCATGGAA GTATCATAGC ACTTACTATT GTCGGCTTTA CTTCTTCTAC TTTTGTATAA 120
TCCTTTTTTT TCTTAGGTTC TGTGATTAGA TATTTTTTTT GTGTGGGTTT AGTTGTTAAT 180
CTGATCATGG ATTTGAACGG TGAGTGTAAA GGAGGAGATG GGTTTATTGA TAGAAGCAGA 240
GTCAGGATTT TGCTTTGTGA CAATGATTCC ACGAGTTTGG GAGAGGTTTT TACTCTCCTT 300
TCAGAGTGTT CTTATCAAGT GACTGCAGTG AAATCAGCAA GGCAGGTGAT TGATGCACTT 360
AATGCAGAGG GACCTGATAT CGATATAATA CTGGCTATTG ACAAAGTA 408
<210> 5
<211> 32
<212> DNA
<213>synthetic, 5 ' ends carry out phosphorylation modification, and left side 14bp is the probe sequence for PEDV M gene, right side
18bp is the labeled primer sequence for arabidopsis TOC1 genetic fragment
<400> 2
TCAGATTCAG GGAGGGCGAC TTTGTAGGGA CA 32
<210> 5
<211> 32
<212> DNA
<213>synthetic, left side 14bp is the probe sequence for PEDV M gene, and right side 18bp is for arabidopsis TOC1
The labeled primer sequence of genetic fragment
<400> 3
TACTTTGTCA ATAGCCAGTA TTATATCGAT AT 32
210> 5
<211> 20
<212> DNA
<213>synthetic, for the reverse primer sequences for arabidopsis TOC1 genetic fragment
<400> 4
TTATACAAAA GTAGAAGAAG 20
<210> 5
<211> 20
<212> DNA
<213>synthetic, for the reverse primer sequences for arabidopsis TOC1 genetic fragment
<400> 5
TTTACTCTCC TTTCAGAGTGT。
Claims (5)
1. the detection kit of a Porcine epidemic diarrhea virus, it is characterised in that: this test kit includes 1 wire, with spy
The reporter gene of specific probes, described reporter sequences is as shown in SEQ NO:1.
The detection kit of Porcine epidemic diarrhea virus the most according to claim 1: it is characterized in that: this test kit includes
1 pair of upstream and downstream reverse primer, reverse primer sequences is as shown in SEQ NO:4 and SEQ NO:5.
The detection kit of Porcine epidemic diarrhea virus the most according to claim 1, it is characterised in that: described report base
Cause and reverse primer are configured to PCR and detect reactant liquor, and test kit also includes enzyme liquid, positive control, negative control.
The detection kit of Porcine epidemic diarrhea virus the most according to claim 1, it is characterised in that: described enzyme liquid by
AMV Reverse Transcriptase (Promega) 0.8 μ L, Tfl DNA Polymerase (Promega) 0.6 μ L,
Taq DNA Ligase(NEB) 0.6 μ L composition；
Reactant liquor, by 5 × Reaction Buffe 5 μ L, 10 × Taq DNA Ligase Reaction Buffer 2.5 μ
The random primer 6 aggressiveness 2.0 μ L of l, 25mM, reporter gene 1.0 μ L, 10mM dNTPs 2.5 μ L, 25mM MgSO4 1μL、10μ
M upstream reverse primer 1.0 μ L, 10 μMs of reverse downstream primer 1.0 μ L, H2O 4.0 μ L forms；
Positive control C is Porcine epidemic diarrhea virus cell culture；
Negative control D is sterilizing distilled water.
5. the detection method of the detection kit of a Porcine epidemic diarrhea virus, it is characterised in that: comprise the steps:
Step 1) extracts the nucleic acid of sample to be checked；
Step 2) detection of PEDV mono-step:
A. prepare 25 μ L reaction systems: the nucleic acid 3 μ L of preparation, reactant liquor 20 μ L, enzyme liquid 2 μ L, do negative and positive comparison simultaneously, mixed
Even reactant liquor, of short duration centrifugal collection liquid is at the bottom of pipe；Described testing conditions: carry out 70 DEG C of 7min on PCR cycle instrument successively,
42 DEG C of 30min, 95 DEG C of 3 min, 1 circulation；95 DEG C of 50sec, 60 DEG C of 10 min, 5 circulations；95 DEG C of 50s, 53 DEG C
50s, 72 DEG C of 50s carry out 25 circulations；
Step 3) takes step 2) product 8 μ L carry out electrophoresis detection with the agarose gel of 2%, observe testing result.
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| CN107699635B (en) * | 2017-06-19 | 2021-08-10 | 中国检验检疫科学研究院 | Fluorescent RPA (reverse transcriptase amplification) detection method for porcine epidemic diarrhea virus |
| CN107299156A (en) * | 2017-08-22 | 2017-10-27 | 河北省检验检疫科学技术研究院 | Detect nucleic acid, real-time fluorescence RT RPA kits and the method for Porcine epidemic diarrhea virus |
| CN108531648A (en) * | 2018-04-11 | 2018-09-14 | 四川农业大学 | It is a kind of it is synchronous detection the diarrhoeal virus of 4 boars oligonucleotide chip and its application |
| CN108531648B (en) * | 2018-04-11 | 2022-03-22 | 四川农业大学 | Oligonucleotide chip for synchronously detecting 4 porcine diarrheal viruses and application thereof |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN111172319A (en) * | 2019-12-20 | 2020-05-19 | 福建傲农生物科技集团股份有限公司 | Primer pair for detecting porcine epidemic diarrhea virus, kit and application thereof |
| CN112662821A (en) * | 2021-01-30 | 2021-04-16 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer combination, kit and application of porcine epidemic diarrhea virus M gene |
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