CN109182596A - A kind of the reverse transcription polymerase spiral response isothermal detection reagent and detection method of Porcine epidemic diarrhea virus - Google Patents
A kind of the reverse transcription polymerase spiral response isothermal detection reagent and detection method of Porcine epidemic diarrhea virus Download PDFInfo
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Abstract
The invention belongs to technical field of virus detection, and in particular to a kind of reverse transcription polymerase spiral amplification isothermal detection methods (Reverse transcription polymerase spiral reaction, RT-PSR) of Porcine epidemic diarrhea virus.The present invention provides the Porcine epidemic diarrhea virus polymerase spiral detection methods of complete set, including detection reagent and reaction condition, a pair of of specific primer is synthesized according to gene conserved regions design, including upstream primer S3, downstream primer S4, detection reagent includes following component: 10 × Bst Buffer reaction buffer, dNTPs, MgSO4, Bst archaeal dna polymerase, AMV reverse transcriptase, DEPC water, colour reagent and above-mentioned primer.Detection method provided by the invention has many advantages, such as that high specificity, sensitivity are high, easy to operate, low in cost in application.
Description
Technical field
The invention belongs to technical field of virus detection, and in particular to a kind of reverse transcription polymerase of Porcine epidemic diarrhea virus
Spiral response (reverse transcription polymerase spiral reaction, RT-PSR) isothermal detection reagent
And detection method.
Background technique
Pig epidemic diarrhea (Porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus
Caused by (Porcine epidemic diarrhea virus, PEDV), virion is rounded or oval, belongs to coronal
There is cyst membrane on Viraceae, surface, have radial fine prominent on cyst membrane, curl winding shape in cyst membrane for what nucleocapsid protein and RNA were formed
Structure.PEDV can cause the pig of each age level to infect, and clinical symptoms cause with infectiousness and marcy agent (TGEV)
Symptom it is similar, mainly include diarrhea, vomiting, tight 4, dehydration and weight loss etc., the clinical symptoms of piglet are more serious, out
Now a large amount of dead, the death rate reaches as high as 100%, brings huge economic loss to pig breeding industry.
The outburst of PEDV is reported in Britain first, and CV777 strain is found in Belgium by separation, is considered as within 1978
Cause the arch-criminal of Belgium outburst PED, it was confirmed that be different from the presence of TGEV, in nineteen eighty-two, be proved to be named as PEDV, hereafter
40 years in, PEDV is broken out in succession in numerous pig-raising countries such as Europe, Asia, America, and causes sizable economic loss.?
Asia, China, just about the report of PED, described a kind of disease similar to clinical symptoms, but until 1984 in 1973
The cause of disease of this disease is caused just to be found by separation in year.Before 2010, although PED happens occasionally in China, presentation is distributed
Property and territorial characteristics.High pathogenic PED has been broken out in south China province in 2010, and spreads to the whole nation rapidly, piglet it is dead
The rate of dying is up to 100%.In Japan, first it is detected that PED is in nineteen eighty-two, and in 2013, highly pathogenic PED is quick-fried in Japan
Hair.1992, there is PED in South Korea's report, 2004-2013 PED is controlled in South Korea, does not occur breaking out on a large scale.
But in 2013, there is the outburst of highly pathogenic strain in South Korea.In Asia, other are regional such as: also there are PED in Thailand, Philippine
Relevant report, and broke out highly pathogenic PED before and after successive in 2013.
The prevalence of PED is usually expressed as susceptible to newborn suckling pig, and causes serious consequence, meanwhile, PED also with its
His etiology coinfection, such as: coli-infection, porcine circovirus 2 type TGEV and rotavirus.Newborn piglet weight subtracts
Gently, watery diarrhea, vomiting, dehydration, high mortality, it is the classical symptom of PED that excrement, which has acid smell in yellow-white,.Histologic lesion
Including acute, diffusivity, serious atrophic enteritis, the upper severe edema due to hypofunction of the spleen of the slight vacuolation of superficial epithelial cell and colon, caecum
It is swollen；During acute infection, observe that the cell of vacuolation or bulk cell peel off in the fluff tip of jejunum or entire villus,
The villus of atrophy is usually merged and is covered on above with degeneration or regenerated squamous epithelium, there is apparent inflammation in intrinsic confluent monolayer cells
Property cellular infiltration.Early stage (i.e. incubation period) the infection pig of PEDV infection shows normal villus length, but empty balloon-shaped cell
Necrosis has begun appearance；1-3 days after diarrhea generation, infected pig showed serious villus and shortens；The infection later period (faces
84-120h after bed symptom occurs) lasting meronecrosis causes moderate to the Villus atrophy of severe.
Structural proteins S, E, M, N in PEDV main code 4, the basic structure of these types of albumen composition virion, and
The duplication of virus plays an important role in amplification.It is a variety of non-structural in virus replication, assembling process in addition to structural proteins
Albumen plays an important role simultaneously.The non-structural protein of ORF1a on PEDV and ORF1b coding, polyprotein pp1a and
pp1b.Polyprotein is cut into 16 kinds of mature replicase under the action of a variety of enzymes, these enzymes are commonly referred to as non-structural
Albumen is named as NSP1-NSP16, their major function is the synthesis for participating in RNA, the duplication of virus and virus and host
Between reaction.ORF3 encodes a kind of non-structural auxilin between S gene and E gene, and this albumen is an ion
Channel protein can be improved the release and pathogenicity of the yield effect virion of virus, research shows that missing ORF3 albumen can
Virus virulence can be caused to weaken；Currently on the market in all vaccine strains, there is missing at similar position in ORF3, therefore,
ORF3 can be used as a mark of the strain that tells the men from the boys, and be used for clinical diagnosis and epidemiological survey.
The diagnostic method of PEDV is broadly divided into two classes: etiological diagnosis and serodiagnosis at present；Etiological diagnosis is main
For the nucleic acid and albumen of virus, serodiagnosis is mainly for the antibody generated after immune response.In recent years, since height causes a disease
Property PEDV the whole world outburst, more fast and accurately detection method be always people pursue direction；Meanwhile PEDV can be same
A variety of disease co-infections, and caused clinical symptoms are similar, are difficult to differentiate between by common means, so, laboratory diagnosis
Technology is particularly important.
Summary of the invention
For overcome common detection efficiency it is low, it is difficult operation, defect at high cost, the purpose of the present invention is to provide boar streams
The reverse transcription polymerase spiral response isothermal detection reagent and detection method of row diarrhea virus, are detected using this method
When, detection time is short, and specificity and sensibility are high, and isothermal reaction does not need specific apparatus, easy to operate and at low cost.
The first purpose of the invention is to provide the reverse transcription polymerase spiral reverses for detecting Porcine epidemic diarrhea virus
Amplimer group is answered, the primer sets are made of following primer:
Upstream primer S1:5 '-tattatgttggcagcgcgtt-3 '；
Downstream primer S2:5 '-tgccgtcataataagctgct-3 '；
Upstream primer S3:5 '-acgaattcgtacatagaagtatag-tattatgttggcagcgcgtt-3 '；
Downstream primer S4:5 '-gatatgaagatacatgcttaagca-tgccgtcataataagctgct-3 '；
A second object of the present invention is to provide a kind of amplification of the reverse transcription polymerase spiral of Porcine epidemic diarrhea virus is anti-
Isothermal detection reagent is answered, the reagent includes primer sets described in claim 1.
Preferably, the reagent also comprises the following components: 10 × Bst Buffer reaction buffer, dNTPs, Mg2+、
Bst archaeal dna polymerase, AMV reverse transcriptase, DEPC water, template ribonucleic acid and colour reagent.
Preferably, Mg in the reagent2+Concentration be 6mM.
Preferably, the concentration of dNTPs is 1.5mM in the reagent；Preferably, colour reagent is phenol in the reagent
Red and cresol red mixed indicator.
Preferably, the reagent further includes positive control.
Third object of the present invention is to provide a kind of amplification of the reverse transcription polymerase spiral of Porcine epidemic diarrhea virus is anti-
Isothermal detection kit is answered, the kit includes above-mentioned reverse transcription polymerase spiral amplified reaction isothermal detection reagent.
Fourth object of the present invention be to provide above-mentioned isothermal detection reagent or isothermal detection kit it is following it is any in
Application:
(1) detection not for the purpose of the diagnosing and treating of disease or auxiliary detection Porcine epidemic diarrhea virus；
(2) product for detecting or assisting detection Porcine epidemic diarrhea virus is prepared；
(3) whether the detection not for the purpose of the diagnosing and treating of disease or auxiliary detection tested animal sample infect pig stream
Row diarrhea virus；
(4) product for detecting or assisting detection tested animal sample whether to infect Porcine epidemic diarrhea virus is prepared；
(5) whether the detection virus to be measured not for the purpose of the diagnosing and treating of disease is Porcine epidemic diarrhea virus；
(6) prepare for detect or assist to detect virus to be measured whether be Porcine epidemic diarrhea virus product.
Fifth object of the present invention is to provide a kind of detections or auxiliary detection tested animal sample whether to infect pig prevalence
The method of property diarrhea virus, this method is not for the purpose of the diagnosing and treating of disease, comprising the following steps:
(1) geneome RNA is extracted from tested animal sample as template, using above-mentioned isothermal detection reagent or above-mentioned
Isothermal detection kit carry out reverse transcription polymerase spiral response, obtain amplified production；Preferably, the polymerase spiral
The reaction condition of reaction is 42 DEG C of constant temperature and acts on 0.5 hour that subsequent 62 DEG C act on 1 hour；
(2) determine whether tested animal sample infects Porcine epidemic diarrhea virus according to following either method:
A, orange-yellow then tested animal sample infection Porcine epidemic diarrhea virus is presented in amplified production；Amplified production presents purple
It is red then tested animal sample is uninfected by Porcine epidemic diarrhea virus；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then tested animal sample infects
Porcine epidemic diarrhea virus；Only primer dimer shadow band, then tested animal sample is uninfected by Porcine epidemic diarrhea virus.
It is Porcine epidemic diarrhea virus sixth object of the present invention is to provide a kind of detection or auxiliary detection virus to be measured
Method, this method is not for the purpose of the diagnosing and treating of disease, comprising the following steps:
(1) geneome RNA is extracted from virus to be measured as template, using above-mentioned isothermal detection reagent or above-mentioned etc.
Warm detection kit carries out reverse transcription polymerase spiral response, obtains amplified production；Preferably, the polymerase spiral response
Reaction condition be 42 DEG C of constant temperature act on 0.5 hour, it is subsequent 62 DEG C act on 1 hour；
(2) determine whether virus to be measured is Porcine epidemic diarrhea virus according to following either method:
A, amplified production is presented orange-yellow, and virus to be measured is Porcine epidemic diarrhea virus；Aubergine is presented then in amplified production
Virus to be measured is not Porcine epidemic diarrhea virus；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then tested animal virus is pig
Epidemic diarrhea virus；Only primer dimer shadow band, then virus to be measured is not Porcine epidemic diarrhea virus.
Beneficial effects of the present invention are as follows:
1. the present invention uses two pairs of specific primers, two identification regions, relatively existing detection method is easy and special
Property it is high.
2. the reverse transcription polymerase spiral response isothermal detection architecture of present invention detection Porcine epidemic diarrhea virus has height
Sensitivity: it is used as reaction template after the Porcine epidemic diarrhea virus RNA extracted is carried out 10 times of progressive dilutions, with same mould
Plate concentration is detected with regular-PCR method, the results showed that reverse transcription polymerase spiral response method is expanded than conventional RT-PCR
Method it is at least 10 times high.It can be seen that this method has higher sensitivity.
The reverse transcription polymerase spiral response method that the present invention detects Porcine epidemic diarrhea virus has high specific: will face
Common avian viral diseases on bed: (pig passes by CSFV (swine fever) and PRRSV (porcine reproductive and respiratory syndrome virus), TGEV
Metachromia gastroenteritis), PRV (porcine pseudorabies), 2 type of pig annulus (PCV2), the tiny PPV of pig and Porcine epidemic diarrhea virus (PEDV)
It respectively as test object, is detected with method of the invention, only PEDV is the positive, has high specific.
The reverse transcription polymerase spiral response method that the present invention detects Porcine epidemic diarrhea virus has rapidity: relatively general
The reaction time of logical RT-PCR reaction a few hours and detection time, detection method only need constant temperature lower 1.5 hours i.e.
Achievable entire reaction and result judgement.
The reverse transcription polymerase spiral response that the present invention detects Porcine epidemic diarrhea virus has operability: relative to normal
RT-PCR is advised, Porcine epidemic diarrhea virus polymerase spiral response does not need expensive PCR instrument, it is only necessary to a simple constant temperature
Reaction can be completed in water-bath；And result directly can visually determine, without specific apparatus such as gel electrophoresises, therefore the present invention is anti-
Answer system that there is stronger operability.
3. detection method is easy to operate, low in cost, can be widely applied.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is PEDV ORF3 gene conserved region RT-PSR amplification figure.
Fig. 2 is the color change results figure that RT-PSR adds nucleic acid dye.
Fig. 3 is PEDV RT-PSR atopic Test Drawing.
Fig. 4 is the digestion qualification result figure of PEDV PSR reaction product.
Fig. 5 is PEDV PSR reaction sensitivity Test Drawing.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Embodiment 1
1, reagent associated materials
Porcine epidemic diarrhea virus is that laboratory separation saves strain.Virus genom DNA/RNA extracts reaction system (north
Jing Quanshijin Bioisystech Co., Ltd), Betaine (SIGMA company), BstDNA polymerase and 10 × Bst Buffer react
Buffer (Biolabs Inc company), dNTP (clontech company), Mg2+And DL2000 (TAKARA company).Colour reagent is
0.025mM is phenol red and the dyed blended liquid of 0.08mM cresol red, is purchased from Solarbio company.
2, the design and synthesis of PSR primer
With reference to the Porcine epidemic diarrhea virus sequence delivered at present, for the area conservative region ORF3 of PEDV Tobamovirus
Domain separately designs 2 primers for RT-PCR control and reverse transcription, packet using Photographing On-line software Primer Premier 5.0
Include S1 and S2 (S1, S2 primer are connected with the conservative sequence of reverse complemental respectively, in favor of cyclization and strand replacement reaction into
Row) and 2 primers (S3 and S4) for spiral expand, S3 and S4 primer 5 ' hold reverse complementary sequence by being adjusted in plant gene
Transformation is taken, no any animal gene matches sequence, and includes GAATTC (EcoR I restriction enzyme site sequence) conducive to following amplification production
The digestion of object identifies that sequence difference is as follows:
Upstream primer S1:5 '-tattatgttggcagcgcgtt-3 '；
Downstream primer S2:5 '-tgccgtcataataagctgct-3 '；
Upstream primer S3:5 '-acgaattcgtacatagaagtatag-tattatgttggcagcgcgtt-3 '；
Downstream primer S4:5 '-gatatgaagatacatgcttaagca-tgccgtcataataagctgct-3 '；
Wherein: GAATTC (EcoR I restriction enzyme site sequence).
The primer that design is completed is synthesized by Beijing AudioCodes bio-engineering corporation, and the primer after synthesis is diluted to DEPC water
10mmol/mL solution, -20 DEG C of preservations.
3, viral genome is extracted
Reaction system is extracted using virus genom DNA/RNA of Beijing Quanshijin Biotechnology Co., Ltd, is extracted thin
Born of the same parents cultivate Porcine epidemic diarrhea virus liquid, the doubtful infected animal tissue sample for having Porcine epidemic diarrhea virus to infect, specificity
Compare virus genome RNA/DNA (i.e. positive control) of viral sample.
4, the foundation of Porcine epidemic diarrhea virus RT-PSR reaction system
Porcine epidemic diarrhea virus reverse transcription isothermal detection reagent includes following component:
Wherein, positive control is the geneome RNA of Porcine epidemic diarrhea virus sample.
Colour reagent is phenol red and cresol red mixed indicator.
Be placed in after said components are mixed in PCR instrument or in water-bath 42 DEG C of constant temperature act on 0.5 hour, subsequent 62 DEG C
Effect 1 hour；It after reaction, can be by amplified production 2% Ago-Gel (ethidium bromide containing 0.5 μ g/mL) electrophoresis
Detection, deposition condition 80V, 30min；The discoloration of its color can also be directly visually observed, the two determines that result is consistent.It visually observes
When, positive findings are shown as orange-yellow, and negative findings are shown as aubergine.
Electrophoresis detection result is as shown in Figure 1, electrophoretic band is consistent with the theoretical results, the expansion of negative control in scalariform distribution
Increase result and there was only primer dimer appearance.The color change of Fig. 2 respectively illustrates the visually observed property of amplification, natural light
Lower positive pipe is orange-yellow, and negative tube keeps aubergine.
Fig. 1 is PEDV ORF3 gene conserved region RT-PSR amplification figure.Wherein M:DNA molecular weight standard DL2000；1:PEDV
Strain amplification；2: negative control.
Fig. 2 is color change results (this dyestuff is addition before reaction) figure of RT-PSR addition nucleic acid dye.Wherein 1-6 is orange
Color, 7 be aubergine.Orange-yellow is the positive, and aubergine is negative control.
5, the optimization of Porcine epidemic diarrhea virus RT-PSR detection method condition
The optimization of 5.1 reaction time
Reaction system is configured, 3 repetitions are arranged in the sample of each time conditions, after 42 DEG C of constant temperature act on 0.5 hour, respectively
It is taken out after reacting 40min, 50min, 60min, 70min in thermostat water bath, after reaction product is all taken out, respectively takes 5 μ L
Product detects reaction product with 2% Ago-Gel (ethidium bromide containing 0.5 μ g/mL) electrophoresis.
The optimization of 5.2 reaction temperatures
Reaction system is configured, each temperature Conditions Sample is arranged 3 repetitions and exists respectively after 42 DEG C of constant temperature act on 0.5 hour
60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, react 1 hour in 65 DEG C of five reaction temperatures after, respectively take 5 μ L products after reaction
Carry out 2% Ago-Gel (ethidium bromide containing 0.5 μ g/mL) electrophoresis detection.
The optimization of each important component concentration in 5.3 reactions
In 25 μ L reaction systems, enzyme unit needed for primer concentration, reaction and template used concentration are constant, change component
MgSO4Concentration, grope MgSO4Influence of the concentration to amplification efficiency, additional amount be followed successively by for 7mM, 6mM, 5mM, 4mM, 3mM,
3 repetitions are arranged in 2mM, 1mM, 0mM, each concentration gradient.After reaction to all concentration gradients, it is solidifying to carry out 2% agarose
Gel electrophoresis detection；The reaction system optimization of different dNTPs concentration is set, gradient be set as 1.8mM, 1.5mM, 1.2mM,
0.9mM, 0.6mM, all grouping reactions all set negative control using deionized water as template, and 3 weights are arranged in each concentration gradient
It is multiple.After reaction to all concentration gradients, 2% agarose gel electrophoresis detection is carried out.
6, the optimum results of Porcine epidemic diarrhea virus PSR isothermal detection methods
6.1 reaction time optimum results: when different between when reacted, it is more and more brighter electrophoretic band to be extended with the time
It is aobvious, when reacting 60min in thermostat water bath, as a result most preferably.
6.2 reaction temperature optimum results: when reaction temperature difference, it may appear that electrophoretic band luminance difference, at 62 DEG C
When reaction, effect is best.
The optimum results of each important component concentration in 6.3 reactions:
Work as MgSO4When concentration difference, it may appear that electrophoretic band luminance difference works as MgSO4When concentration is 6mM, effect is best.
When dNTPs concentration difference, it may appear that electrophoretic band luminance difference, when dNTPs concentration is 1.5mM, effect is most
It is good.
6.4 optimization systems and condition
By the optimization of above-mentioned condition, the last optimization of Porcine epidemic diarrhea virus reverse transcription polymerase spiral response method
System are as follows:
Porcine epidemic diarrhea virus reverse transcription polymerase spiral response detection reagent includes following component:
The reaction condition of one spiral methods of polymerase is 42 DEG C of constant temperature and acts on 0.5 hour that subsequent 62 DEG C act on 1 hour.
6.5 Porcine epidemic diarrhea virus reverse transcription polymerase spiral response detection kits: the pig stream of 25 μ L detection architectures
Row diarrhea virus reverse transcription polymerase spiral response reaction system includes following component:
7, the specific test of PSR
With clinically common porcine viral disease: CSFV (swine fever) and PRRSV (porcine reproductive and respiratory syndrome disease
Poison), TGEV (transmissible gastroenteritis of swine), PRV (porcine pseudorabies), 2 type of pig annulus (PCV2) and pig tiny (PPV) be as detecting
Object carries out PSR detection with PEDV primer of the invention, to verify the specificity of its reaction, and positive control is arranged, as a result joins
See Fig. 3.
The reaction system of PSR detection includes following component:
Wherein, positive control is Porcine epidemic diarrhea virus template DNA, to extract reactant using viral DNA/RNA
It is the Porcine epidemic diarrhea virus genomic DNA extracted.
Amplification shows that scalariform band occurs in only positive control, other 6 kinds of viruses do not occur, illustrate with other diseases
Poison does not have positive amplification when being used as template, the PEDV PSR primer that the present invention designs has the specificity of height.
Fig. 3 is PEDV RT-PSR atopic Test Drawing.Wherein M:DNA molecular criteria amount standard DL2000；Swimming lane 1
~7 be respectively PEDV RT-PSR specific primer detection positive control (PEDV), CSFV and PRRSV, TGEV, PRV, PCV2 and
PPV's as a result, 8 be negative control.
The digestion of 7.1 amplified productions is identified
For the specificity for further verifying amplified reaction, product carries out digestion identification after RT-PSR is reacted, anti-in 20 μ L
It answers and following components is added in system, positive PSRP product is digested with EcoR I.
After above-mentioned reaction product is mixed, 37 DEG C of reaction 2h take out 5 μ L products, (are contained with 2% Ago-Gel after reaction
The ethidium bromide of 0.5 μ g/mL) digestion products are detected.
As a result: in theoretical calculation, after restriction endonuclease EcoR I digestion, should become three sizes is respectively PEDV amplified production
The master tape of 50bp, 220bp and 260bp.Result is consistent completely with theoretical value in Fig. 4, illustrates the detection method tool that the present invention establishes
There is good specificity.
Fig. 4 is the digestion qualification result figure of PEDV PSR reaction product.Wherein M:DNA molecular criteria amount standard DL2000；
1:PEDV PSR final product；2: restriction endonuclease EcoR I postdigestive result.
8, the sensitivity tests of PEDV
Take 10 times of the virus liquid DNA product extracted progress is progressive to be diluted to 10-7, and it is divided into two parts, it is a part of with excellent
PSR method detection after change, directly visually observes after developing solution is added, measures the sensibility of this method；Another part is conventional
PCR method detection, the two testing result is compared, and is schemed referring to a and b of Fig. 5, to verify the sensibility of its reaction.
The result shows that: RT-PSR method is at least 10 times higher than the method that conventional RT-PCR expands.It can be seen that of the invention
RT-PSR method has higher sensibility.
Fig. 5 is PEDV PSR reaction sensitivity Test Drawing, wherein M:DNA molecular criteria amount standard DL2000；Wherein, a schemes
For regular-PCR method, b figure is PSR method of the invention.A, 1~6 respectively template is diluted to 10 in b figure-1、10-2、10-3、
10-4、10-5、10-6, 7 be negative control.
9, the accordance of RT-PSR practical application
The tissue disease sample of the doubtful Porcine epidemic diarrhea virus of clinical acquisitions extracts sample gene group, is established using the present invention
PSR detection method and conventional PCR and fluorescence quantifying PCR method detected, by three kinds of method practical applications
Testing result compares, and verifies the accordance of RT-PSR.
The result shows that: utilize Porcine epidemic diarrhea virus reverse transcription polymerase isothermal spiral response reaction system of the invention
The sample of acquisition is detected with Standard PCR and quantitative fluorescent PCR, it is found that the recall rate of RT-PSR of the invention is higher than and passes
The PCR method of system, RT-PCR more close with fluorescence quantifying PCR method recall rate and traditional and fluorescence quantitative RT-RCR inspection
Positive sample out can be detected by PSR method of the invention.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (10)
1. the reverse transcription polymerase spiral response isothermal duplication primer sets for detecting Porcine epidemic diarrhea virus, feature exist
In: the primer sets are made of following primer:
Upstream primer S1:5 '-tattatgttggcagcgcgtt-3 '；
Downstream primer S2:5 '-tgccgtcataataagctgct-3 '；
Upstream primer S3:5 '-acgaattcgtacatagaagtatag-tattatgttggcagcgcgtt-3 '；
Downstream primer S4:5 '-gatatgaagatacatgcttaagca-tgccgtcataataagctgct-3 '.
2. a kind of reverse transcription polymerase spiral amplified reaction isothermal detection reagent of Porcine epidemic diarrhea virus, it is characterised in that:
The reagent includes primer sets described in claim 1.
3. reagent according to claim 2, it is characterised in that: the reagent also comprises the following components: 10 × Bst
Buffer reaction buffer, dNTPs, Mg2+, Bst archaeal dna polymerase, AMV reverse transcriptase, DEPC water, template ribonucleic acid and colour developing examination
Agent.
4. reagent according to claim 3, it is characterised in that: Mg in the reagent2+Concentration be 6mM.
5. reagent according to claim 3, it is characterised in that: the concentration of dNTPs is 1.5mM in the reagent；As excellent
It selects, colour reagent is phenol red and cresol red mixed indicator in the reagent.
6. reagent according to claim 3, it is characterised in that: the reagent further includes positive control.
7. a kind of reverse transcription polymerase spiral amplified reaction isothermal detection kit of Porcine epidemic diarrhea virus, feature exist
In: the kit includes the described in any item reverse transcription polymerase spiral amplified reaction isothermal detection reagents of claim 2-6.
8. the described in any item isothermal detection reagents of claim 2-6 or isothermal detection kit as claimed in claim 7 are such as
Under it is any in application:
(1) detection not for the purpose of the diagnosing and treating of disease or auxiliary detection Porcine epidemic diarrhea virus；
(2) product for detecting or assisting detection Porcine epidemic diarrhea virus is prepared；
(3) whether the detection not for the purpose of the diagnosing and treating of disease or auxiliary detection tested animal sample infect pig popularity
Diarrhea virus；
(4) product for detecting or assisting detection tested animal sample whether to infect Porcine epidemic diarrhea virus is prepared；
(5) whether the detection virus to be measured not for the purpose of the diagnosing and treating of disease is Porcine epidemic diarrhea virus；
(6) prepare for detect or assist to detect virus to be measured whether be Porcine epidemic diarrhea virus product.
9. whether a kind of detection or auxiliary detection tested animal sample infect the method for Porcine epidemic diarrhea virus, this method not with
For the purpose of the diagnosing and treating of disease, it is characterised in that: the following steps are included:
(1) geneome RNA is extracted from tested animal sample as template, using the described in any item isothermals of claim 2-6
Detection reagent or isothermal detection kit as claimed in claim 7 carry out reverse transcription polymerase spiral response, obtain amplified production；
It is acted on 0.5 hour preferably, the reaction condition of the polymerase spiral response is 42 DEG C of constant temperature, subsequent 62 DEG C act on 1 hour；
(2) determine whether tested animal sample infects Porcine epidemic diarrhea virus according to following either method:
A, orange-yellow then tested animal sample infection Porcine epidemic diarrhea virus is presented in amplified production；Aubergine is presented in amplified production
Then tested animal sample is uninfected by Porcine epidemic diarrhea virus；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then tested animal sample infection pig stream
Row diarrhea virus；Only primer dimer shadow band, then tested animal sample is uninfected by Porcine epidemic diarrhea virus.
10. a kind of detection or auxiliary detection virus to be measured are the method for Porcine epidemic diarrhea virus, this method not examining with disease
For the purpose of disconnected and treatment, it is characterised in that: the following steps are included:
(1) geneome RNA is extracted from virus to be measured as template, is detected using the described in any item isothermals of claim 2-6
Reagent or isothermal detection kit as claimed in claim 7 carry out reverse transcription polymerase spiral response, obtain amplified production；As
It is acted on 0.5 hour it is preferred that the reaction condition of the polymerase spiral response is 42 DEG C of constant temperature, subsequent 62 DEG C act on 1 hour；
(2) determine whether virus to be measured is Porcine epidemic diarrhea virus according to following either method:
A, amplified production is presented orange-yellow, and virus to be measured is Porcine epidemic diarrhea virus；It is then to be measured that aubergine is presented in amplified production
Virus is not Porcine epidemic diarrhea virus；
B, amplified production is subjected to agarose gel electrophoresis, electrophoretic band is distributed in scalariform, then tested animal virus is pig prevalence
Property diarrhea virus；Only primer dimer shadow band, then virus to be measured is not Porcine epidemic diarrhea virus.
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