CN101575650A - Porcine reproductive and respiratory syndrome virus RT-LAMP detection kit and detection method thereof - Google Patents
Porcine reproductive and respiratory syndrome virus RT-LAMP detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to a porcine reproductive and respiratory syndrome virus RT-LAMP detection kit and a detection method thereof. Primers required by PRSSV RT-LAMP reaction system are designed according to the sequence of porcine reproductive and respiratory syndrome virus (PRSSV) published by GenBank; PRSSV virus RNA is extracted with the virus RNA extraction reagent (LBBII-RNA) designed and prepared by the inventor, the PRSSV RT-LAMP reaction system established in the invention is utilized for detection, and color developing agent is added after the reaction to judge the result; the result shows that the PRSSV virus RNA obtains efficient specific amplification after the reaction is conducted for 45 minutes at the temperature of 63 DEG C; and then, quick detection of porcine reproductive and respiratory syndrome virus American classical strain and NSP2 variant strain (highly pathogenic porcine reproductive and respiratory syndrome virus strain) is conducted by SpuI enzyme cutting. Compared with the prior art, the invention has quick detection, high sensitivity, low reaction cost, convenient and fast operation, which is capable of differentiating American classical strain and NSP2 variant strain (highly pathogenic porcine reproductive and respiratory syndrome virus strain) and meets the requirement of multi-level detection.
Description
Technical field
The present invention relates to porcine reproductive and respiratory syndrome virus RT-LAMP detection kit and detection method thereof, belong to the eqpidemic disease diagnostic techniques in the veterinary biologics field.
Background technology
Porcine reproductive and respiratory syndrome, claim blue otopathy again, be by porcine reproductive and respiratory syndrome virus (PorcineReproductive and Respiratory Syndrome Virus, PRRSV) causing a kind of is the transmissible disease of feature with sow breeding difficulty and piglet respiratory tract disease, is pig industry is threatened bigger transmissible disease.
PRRSV belongs to shell type virales, Arteriviridae.This togavirus is the underlying stock single strand RNA virus, and its mrna length is 15kb.Present Chinese popular PRRSV virus stain is the american type PRRSV strain (high-pathogenicity blue ear disease strain) of american type and NSP2 variation.Detection method that at present should virus has RT-PCR method, cellular segregation to identify and method such as ELASA.But in actual PRRSV testing, the following problem that the detection technique means that adopted exist: common PCR method: be easy to generate false positive, template quality required high; Common serological method: susceptibility is not enough, and complex operation is not suitable for the Detection of antigen work of requirements at the higher level; The cellular segregation culture identification: the cycle is long, to the requirement for experiment condition harshness.
The isothermal amplification (LAMP) of ring mediation is a kind of novel nucleic acids amplification technique (Notomi by inventions such as T.Notomi, T., et al., Loop-mediated isothermal amplification of DNA.Nucleic Acids Res.2000,28, e63.), this technology relies on primer and a kind of archaeal dna polymerase with strand displacement characteristic of 4 special designs, can be efficiently under isothermal condition, high amplified target sequence specifically.In recent years, this technology is widely used in pathogen detection abroad.People (2007) such as Masaki Imai at four kinds of cordiale zymic ITS sequences Design the LAMP Auele Specific Primer, set up LAMP detection architecture efficiently.LAMP can also detect other and human relevant virus, as viral hemorrhagic septicemia (VHS), cytomegalovirus (CMV), Ebola virus (EBOV), chronic burkitt's lymphoma virus (EBV), rainbow virus, human herpes virus type 8, hematopoietic tissue necrosis virus (IHHNV), tomato spotted wilf virus, tomato yellow leaf curl virus etc.There is no both at home and abroad at present and be useful on RT-LAMP test kit and the application of LAMP method in porcine reproductive and respiratory syndrome virus detects that detects porcine reproductive and respiratory syndrome virus, also do not see the report that has at PRRSV american type classical strains and high-pathogenicity blue ear disease virus (the american type PRRSV of NSP2 variation) the effective LAMP detection method of distinguishing of strain.
The problem that porcine reproductive and respiratory syndrome virus RT-LAMP detection kit of the present invention and detection method thereof have avoided above-mentioned detection means to exist, compare other detection means, the PRRSV RT-LAMP method of setting up has high specificity, susceptibility height, short, the simple to operation characteristics of sense cycle, has effectively solved the problem of the other technologies means existence of using in testing.
Summary of the invention
The objective of the invention is to set up and detect porcine reproductive and respiratory syndrome virus, particularly can be used for distinguishing the RT-LAMP detection method of classical strain of PRRSV american type and NSP2 variant, and a kind of RT-LAMP test kit that is used for above purpose is provided.
Technical scheme of the present invention
Principle of the present invention and technological line
The present invention will set up at PRRSV american type classical strains and PRRSV NSP2 variant (high-pathogenicity blue ear disease strain) loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) detection method, present method has many primers and increases simultaneously, and formed the ring texture that has the primer function at two ends, it is highly sensitive that this many primers combination and the principle that can produce primer have certainly had it, characteristics such as high specificity, because RT and LAMP are reflected at having simplified operation steps in the pipe simultaneously, simultaneously because comprise the precipitation of a large amount of nucleic acid and magnesium pyrophosphate in the product of RT-LAMP reaction, can develop the color with fluorescent agent and verify, and the purpose fragment in the reactant contains restriction enzyme site, can cut evaluation with enzyme, ways such as agarose gel electrophoresis are come the confirmatory reaction result, are applicable to the use of testing under the various experiment conditions.
Method of the present invention is specifically implemented
The foundation and the checking of 1 pig PRSSV RT-LAMP detection method
(1) the reaction preparation of reagent
1) design of primers, screening PRRSV RT-LAMP design of primers are template with american type PRRSV strain (high-pathogenicity blue ear disease GD strain, Beijing strain) gene order of the PRRSV (America strain VR-2332) that screens the classics of announcing according to GenBank, other 4 strain american type strains and NSP2 variation, RT-LAMP detects primer in the both sides design of high-pathogenicity blue ear disease virus N SP2 variant disappearance 90bp nucleotide sequence, utilize LAMP Tubidimeter analyser that the bearing reaction of different primers is screened, by wherein selecting two pairs of primers:
F3：CAGATCCGATTGCGGCAG
B3：TTCTACGCGGTGCAGGAA
FIP：CATCGGCTCGGATGGTGTCGATGGGCGACAATGTCCCTA
BIP：ACCCATGAGTGAGCCCGTACTCGACCCACTCAAAGGTTTCA
2) preparation of reagent in the RT-LAMP reaction system (50 reacting weights)
1. primer mixed solution (PM): get 100pmol/ μ l F3 2.5 μ l, 100pmol/ μ l B3 2.5 μ l, 100pmol/ μ l FIP20 μ l, add sterilization deionized water 5 μ l after 100pmol/ μ l BIP 20 μ l P mix, total system 50 μ l.Each reaction need be got 1 μ lPB.
2. react buffer mixture (RM): get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ ldNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed molten to 625 μ l.Each reaction need be got 12.5 μ l RB.
3. reaction enzymes mixed solution (EM): get 16U/ μ l Bst large fragment DNA polysaccharase 24 μ l, 400U/ μ l AMV ThermoScript II 24 μ l, 1 μ mol/ μ l DTT, 2 μ l.Each reaction needed is got 1 μ l EB.
4. developer:, get 1 μ lSYBRgreenI (available from Invitrogen company) and be dissolved in 49 μ l PCR level water.Each reaction adds 1 μ l.
5. restriction enzyme Spu I:Spu I 1000u/ml (available from NEB company).
(2) preparation of sample RNA
1) RNA extracts reagent (LBB II-RNA)
RA: get 1%2-mercaptoethanol 0.60ml, 1%Tris-HCL (pH 8.0) 0.60ml, 10mmol/ml EDTA 1ml and be dissolved in the 5.0ml sterilization distilled water.
RB:6mmol/ml guanidinium isothiocyanate 35ml.
RC: dehydrated alcohol 15ml.
RD:100u/ μ l DNAse A 20 μ l are dissolved in 10ml DEPC H
2O.
2) extraction of sample RNA
Get 100 μ l cell cultures (or serum sample, organize ground sample), behind multigelation 3 times, operate as follows:
Add sample equal-volume RA in the sample, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places new pipe;
The RB solution that in 1, adds 3 times in the supernatant liquor, vortex or concuss 90s leave standstill 3min;
The RC solution that adds 1/3 volume, vortex 1min, the centrifugal 1min of 12000g.Abandoning supernatant; Sample drying behind the placement 5min adds 11 μ l RD again, and dissolution precipitation is sample RNA, as the RNA template of amplification usefulness.
(3) sample detection
1) the first step (reaction system is 20 μ l)
1. prepare RT-LAMP reaction solution: RM 12.5 μ l, EM 1.0 μ l, PM 1.0 μ l;
2. getting 5.5 μ l sample RNA is added in the RT-LAMP reaction solution that 1. item is prepared;
3. mixed back acts on 45min in 60～65 ℃ of thermostat water baths.
4. the result judges: the reaction back adds the developer of 1 μ L in PRSSV RT-LAMP reaction product, and reaction finishes back visual inspection reaction solution colour-change.The reaction solution of negative reaction presents orange, and the reaction solution of positive is green (seeing accompanying drawing 1).
2) second step, positive and need further to determine to carry out when its product is the PRRSV american type classical strains or the high-pathogenicity blue ear disease strain of PRRSV NSP2 variation as the first step result:
1. take out adding SpuI enzyme system 9 μ L among the 1 μ L in the positive products of PRRSV RT-LAMP reaction, totally 10 μ L systems are in 37 ℃ of warm bath effect 1h.
2. prepare 1% sepharose.
3. enzyme is cut in the gel of product described in 2. and is carried out electrophoresis in will be 1..Use Marker 2000plus as the electrophoresis reference standard.
4. observations:
Declaring evaluation criteria is: lane shown in the accompanying drawing 81 occurring is that porcine reproductive and respiratory syndrome virus american type NSP2 variant (high-pathogenicity blue ear disease strain) RT-LAMP product is through SpuI enzyme effect rear electrophoresis result; Lane2 is that classic America strain RT-LAMP product is through SpuI enzyme effect rear electrophoresis result shown in the appearance accompanying drawing 8.
(4) checking of reaction result
1) the electrophoresis qualification result of RT-LAMP product is got 10 μ L PRRSV RT-LAMP amplified productions electrophoresis detection on 1% sepharose, 100U 30min observations in Biohazard Safety Equipment.The RNA sample RT-LAMP of PRRSV american type classical strains (VR-2332) and NSP2 variant detects, and the result all is positive, and the stepped distribution of RT-LAMP amplified production amplifying nucleic acid electrophoretic band ( lane 2,3,4,5) conforms to notional result.Negative (lane 1) contrast does not see that (see figure 2) appears in the specificity product.
2) enzyme of RT-LAMP product is cut evaluation and is adopted restriction enzyme SpuI to carry out enzyme the RT-LAMP product to cut evaluation.By specification preparation reaction solution behind 37 ℃ of effect 1h, is got 10 μ L reaction product electrophoresis and observations (as Fig. 3) in 1% sepharose.Lane 2 is that VR-2332 strain RT-LAMP amplified production is after restriction endonuclease SpuI digestion, formation is about the electrophoresis banding pattern of 160bp and 120bp based on band, and high-pathogenicity blue ear disease virus (GD strain) amplified production of NSP2 variation is unaffected, conforms to expected results.
3) the visual qualification result of RT-LAMP product adds the developer of 1 μ L, visual inspection reaction solution colour-change in PRSSV RT-LAMP reaction product after the RT-LAMP reaction finishes.The reaction solution of negative reaction presents orange, and the reaction solution of positive is green (seeing accompanying drawing 4).
4) viral nucleic acids such as PRRSV RT-LAMP specificity test extracting PRSSV VR-2332 strain, GD strain according to the PRRSV RT-LAMP detection method of being set up, carry out the specificity test.Extract Pestivirus suis, the sick virus of porcine influenza, pig circular ring virus, the tiny sick virus control sample nucleic acid of pig respectively, with porcine reproductive and respiratory syndrome virus RNA as positive control, the water of handling with DEPC is as negative control, detect the specificity of the RT-LAMP method of setting up, positive control and negative control are all set up as a result, PRRSV is positive, and other test sample is negative (seeing accompanying drawing 5), and the result shows: this method has specificity to PRRSV.
5) (titre is 100TCID in PRRSV RT-LAMP sensitivity test extraction PRRSV GD strain
50) RNA, carry out 10 times of serial dilutions (1～1.0 * 10
-6 Deng 7 extent of dilution), use the RT-LAMP detection method of being set up to carry out sensitivity test, reaction product is verified with gel electrophoresis.Electrophoresis result shows that along with the reduction of template concentrations, the brightness of amplified production band is not remarkable from difference, to 10
-6Do not have amplified production not have band during extent of dilution yet and (see figure 6) occurs.
6) PRRSV RT-LAMP field application result is gathered the porcine blood serum and the morbidity swine disease material of 30 parts of PRRS hotspots, and cultivate with the Marc145 cellular segregation, extract total RNA of 30 duplicate samples simultaneously, detect simultaneously with RT-LAMP detection method and the RT-PCR method set up, and cut the product of RT-LAMP with the SpuI enzyme.The result shows in 30 duplicate samples that it is the PRRSV positive that RT-LAMP has detected 7 duplicate samples, detect with Marc145 cell cultures, RT-PCR (with the development of Chinese Animal diseases control center specially at the test kit of variant) the identical rate of synthesis result be 100%.Wherein the RT-LAMP product in 5 parts of positive can be cut by the SpuI enzyme, tentatively is defined as the american type classical strains, the results are shown in Table 1.
Table 1.PRRSV RT-LAMP field application experiment positive findings
The preparation of 2 test kits (50 reacting weight/boxes)
(1) preparation of the required reagent LBB II-RNA of extraction sample RNA:
RA: get 1%2-mercaptoethanol 0.6ml, pH 8.0 1%Tris-HCL 0.6ml, 10mmol/ml EDTA 1ml and be dissolved in the 5.0ml sterilization distilled water, be filled in the plastic containers of rnase-free.
RB:6mmol/ml guanidinium isothiocyanate 35ml is filled in the plastic containers of rnase-free.
RC: dehydrated alcohol 15ml is filled in the plastic containers of rnase-free.
RD:100u/ μ l DNAse A 20 μ l are dissolved in 10ml DEPC H
2O is filled in the plastic containers of rnase-free.
(2) the reaction preparation of reagent
1. PM: get 2.5 μ l 100pmol/ μ l F3,2.5 μ l 100pmol/ μ l B3,20 μ l 100pmol/ μ l FIP, add sterilization deionized water 5 μ l after 20 μ l100pmol/ μ l BIP mix, total system 50 μ l are filled in the plastic containers of rnase-free.
2. RM: get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ l dNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed moltenly, be filled in the plastic containers of rnase-free to 625 μ l.
3. EM: get 16U/ μ l Bst large fragment DNA polysaccharase 24 μ l, 400U/ μ l AMV ThermoScript II 24 μ l, 1 μ mol/ μ lDTT, 2 μ l are filled in the plastic containers of rnase-free.
4. developer: get 1 μ lSYBRgreenI (available from Invitrogen company) and be dissolved in 49 μ l PCR level water, be filled in the plastic containers of rnase-free.
5. restriction enzyme SpuI:Spu I 1000u/ml (available from NEB company).
3. the use of test kit
1) use LBBII-RNA to extract sample RNA
Get 100 μ l cell cultures (or serum, organize ground sample), behind multigelation 3 times, operation as follows:
Add sample equal-volume RA in the sample, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places tubule; The RB solution that in supernatant liquor, adds 3 times, vortex or concuss 90s leave standstill 3min; The RC solution that adds 1/3 volume, vortex 1min, the centrifugal 1min of 12000g.Abandoning supernatant; Treat to add 11 μ l RD behind the sample drying, dissolution precipitation is sample RNA.
2) RT-LAMP reaction
1. prepare RT-LAMP reaction solution: RM 12.5 μ l, EM 1.0 μ l, PM 1.0 μ l;
2. get 5.5 μ l sample RNA and be added to 1) the preparation reaction solution in
3. mixed back acts on 45min in 60～65 ℃ of thermostat water baths.
3) result judges: the reaction back adds the developer of 1 μ L, visual inspection reaction solution colour-change in the PRRSV-RT-LAMP reaction product.The reaction solution of negative reaction presents orange, and the reaction solution of positive is green (seeing accompanying drawing 7).
Positive and need further to determine to carry out when its product is the PRRSV american type classical strains or the high-pathogenicity blue ear disease strain of PRRSVNSP2 variation as the first step result:
1. take out adding SpuI enzyme system 9 μ L the 1 μ L from the positive products of PRRSV RT-LAMP reaction, totally 10 μ L systems are in 37 ℃ of warm bath effect 1h.
2. prepare 1% sepharose.
3. enzyme is cut in the gel of product described in 2. and is carried out electrophoresis in will be 1..Use Marker 2000plus as the electrophoresis reference standard.
4. observations:
Declaring evaluation criteria is: lane shown in the accompanying drawing 81 occurring is that porcine reproductive and respiratory syndrome virus american type NSP2 variant (high-pathogenicity blue ear disease strain) RT-LAMP product is through SpuI enzyme effect rear electrophoresis result; Lane2,3 is through SpuI enzyme effect rear electrophoresis result through America type strain RT-LAMP product shown in the appearance accompanying drawing 8.
Description of drawings:
Fig. 1: reaction product adds chromogenic reagent result 1. positive 2. negative samples.
The electrophoresis qualification result M:DL2000 Plus Marker lane 1 of Fig. 2 .RT-LAMP product: negative control lane2,3:PRRSV (VR-2332) lane4,5:PRRSV GD strain
The RT-LAMP amplified production restriction enzyme SpuI restriction enzyme digestion and electrophoresis imaging laneM:DL2000Plus of Fig. 3 PRRSV, lane1:GD strain RT-LAMP product, lane2:VR-2332 strain RT-LAMP product.
Fig. 4: reaction product adds chromogenic reagent result 1,3 positive sample 2,4 negative samples
Fig. 5: PRRSV RT-LAMP specificity test-results 1～3:PRRSV (GD strain) lane 4～6:PRRSV (VR-2332) lane 7:CHLV Lane8:SIV lane9:PCV2 lane 10:PPV
Fig. 6: VR-2332 strain RT-LAMP sensitivity test product gel electrophoresis imaging laneM:DL2000PlusMarker, lane1: negative control, Lane2-9: the viral dilution degree is respectively 10-8,10-7,10-6,10-1,10-2,10-3,10-4,10-5.
Fig. 7: reaction product adds chromogenic reagent result 1 negative sample 2,3,4 and 5 positive samples
Fig. 8 RT-LAMP amplified production SpuI restriction enzyme digestion and electrophoresis imaging results Lane1:SD2 (enzyme is cut feminine gender), Lane2:HN13, lane3:SD1.
Fig. 9: reaction product adds chromogenic reagent result 1,2,3 positive sample 4 negative samples
The present invention compared with prior art, its good effect shows: the present invention relates to a kind of porcine reproductive and respiratory syndrome disease Poison RT-LAMP detection kit and detection method thereof. Porcine reproductive and respiratory syndrome virus according to the GenBank announcement (PRSSV) sequence has designed the required primer of PRSSV RT-LAMP reaction system; Adopt inventor's design and preparation Viral RNA extract reagent and extract the PRSSV viral RNA, the PRSSV RT-LAMP reaction of using the present invention to set up System detects, and adds the developer result of determination after reaction finishes, and the result is presented at 63 ℃ of 45min, PRSSV virus RNA has obtained efficient specific amplification; Recycling SupI enzyme is cut and is carried out porcine reproductive and respiratory syndrome virus american type warp The fast detecting of allusion quotation strain and NSP2 variant. Compare prior art, it is fast that the present invention has detection speed, the strong and energy of sensitivity Distinguish and detect american type classical strains and NSP2 variant, and reaction cost is low, simple operation, be fit to multi-level inspection The survey demand.
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
PRRSV RT-LAMP design of primers and screening
American type PRRSV strain (high-pathogenicity blue ear disease GD strain, Beijing strain) gene order of PRRSV (America strain VR-2332), other 4 strain american type strains and the NSP2 variation of the classics of announcing according to GenBank is a template, lack 90bp nucleotide sequence both sides design RT-LAMP at high-pathogenicity blue ear disease virus N SP2 region of variability and detect primer, utilize LAMP Tubidimeter that the bearing reaction of different primers is screened, by wherein selecting sequence 2 pairs of primers shown below:
F3：CAGATCCGATTGCGGCAG
B3：TTCTACGCGGTGCAGGAA
FIP：CATCGGCTCGGATGGTGTCGATGGGCGACAATGTCCCTA
BIP：ACCCATGAGTGAGCCCGTACTCGACCCACTCAAAGGTTTCA
And determine above primer the primer mixed solution (PrimerMix, PM) each components in proportions:
100pmol/ μ l F3 2.5 μ l, 100pmol/ μ l B3 2.5 μ l, 100pmol/ μ l FIP 20 μ l add sterilization deionized water 5 μ l, total system 50 μ l after 100pmol/ μ l BIP20 μ l mixes.
The preparation of component in the test kit:
Be mixed with various components and divide by the prescription (50 reacting weights) of following various components and install in glass or the plastic small container and use corresponding plug seal:
1.LBBII-RNA this reagent set is made of RA, RB, RC, RD four parts:
RA: get 1%2-mercaptoethanol 0.6ml, pH 8.0 1%Tris-HCL 0.6ml, 10mmol/ml EDTA 1ml and be dissolved in the 5.0ml sterilization distilled water.
RB:6mmol/ml guanidinium isothiocyanate 35ml.
RC: dehydrated alcohol 15ml.
RD:100u/ μ l DNAse A 20 μ l are dissolved in 10ml DEPC H
2O,
2.LAMP reaction reagent
1) PM: get 2.5 μ l 100pmol/ μ l F3,2.5 μ l 100pmol/ μ l B3,20 μ l 100pmol/ μ l FIP, add sterilization deionized water 5 μ l after 20 μ l100pmol/ μ l FIP mix, total system 50 μ l.Each reaction need be got 1 μ l PB.
2) RM: get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ l dNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed molten to 625 μ l.Each reaction need be got 12.5 μ l RB.
3) EM: get 16U/ μ l Bst large fragment DNA polysaccharase 24 μ l, 400U/ μ l AMV ThermoScript II 24 μ l, 1 μ mol/ μ lDTT, 2 μ l.Each reaction needed is got 1 μ l EB.
4) developer: developer constitutes (available from Invitrogen company) by single agents SYBRgreen I fluorescein, gets 1 μ lSYBRgreenI and is dissolved in 49 μ l PCR level water.Each reaction adds 1 μ l.
3. restriction enzyme: SpuI 1000u/ml (available from NEB company).
The use of PPRSV RT-LAMP test kit
1. use LBBII-RNA to extract sample RNA
Get 100 μ l cell cultures (or serum, organize ground sample), behind multigelation 3 times, operation as follows:
Add sample equal-volume RA in the sample, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places tubule; The RB solution that in supernatant liquor, adds 3 times, vortex or concuss 90s leave standstill 3min; The RC solution that adds 1/3 volume, vortex 1min, the centrifugal 1min of 12000g.Abandoning supernatant; Treat to add 11 μ l RD behind the sample drying, dissolution precipitation is sample RNA.
2.PRSSV RT-LAMP reaction
1. prepare the RT-LAMP reaction solution: with RM 12.5 μ l, EM 1.0 μ l, PM 1.0 μ l are added in the reaction tubes;
2. getting 5.5 μ l sample RNA 1. is added in the pipe of the reaction solution of preparation;
3. after mixed reaction tubes is placed 60～65 ℃ of thermostat water bath effect 45min.
3 results judge: the reaction back adds the developer of 1 μ L, visual inspection reaction solution colour-change in the PRRSV-RT-LAMP reaction product.The reaction solution of negative reaction presents orange, and the reaction solution of positive is green (seeing accompanying drawing 9).
When the detected result of embodiment 3 is positive, carry out when further discriminating is the PRRSV american type classical strains or the high-pathogenicity blue ear disease strain of PRRSV NSP2 variation as need:
1. take out adding SpuI enzyme system 9 μ L among the 1 μ L in the positive products of PRRSV RT-LAMP reaction, totally 10 μ L systems are in 37 ℃ of warm bath effect 1h.
2. prepare 1% sepharose.
3. enzyme is cut in the gel of product described in 2. and is carried out electrophoresis in will be 1..Use Marker 2000plus as the electrophoresis reference standard.
4. observations:
Declaring evaluation criteria is: lane shown in the accompanying drawing 81 occurring is that porcine reproductive and respiratory syndrome virus american type NSP2 variant (high-pathogenicity blue ear disease strain) RT-LAMP product is through SpuI enzyme effect rear electrophoresis result; Lane2 shown in the accompanying drawing 8 occurring is that the classical strain RT-LAMP of american type product is through SpuI enzyme effect rear electrophoresis result.
Sequence table
＜110〉China Veterinery Drug Inspection Office
＜120〉porcine reproductive and respiratory syndrome virus LAMP detection kit and detection method thereof
<160>4
<210>1
<211>18
<212>DNA
<213>Forward?Outer?F3
＜223〉artificial sequence
<400>1
CAGATCCGAT?TGCGGCAG 18
<210>2
<211>18
<212>DNA
<213>Reverse?Outer?B3：
＜223〉artificial sequence
<400>2
TTCTACGCGG?TGCAGGAA 18
<210>3
<211>39
<212>DNA
<213>Forward?Inner?FIP：
＜223〉artificial sequence
<400>3
CATCGGCTCG?GATGGTGTCG?ATGGGCGACA?ATGTCCCTA 39
<210>4
<211>41
<212>DNA
<213>Reverse?Inner?BIP
＜223〉artificial sequence
<400>4
ACCCATGAGT?GAGCCCGTAC?TCGACCCACT?CAAAGGTTTCA
Claims (6)
1. RT-LAMP detection kit that detects porcine reproductive and respiratory syndrome virus, its feature comprise extracts RNA reagent LBB II-RNA; RT-LAMP reaction reagent and three components of restriction enzyme Spu I.
2. a kind of RT-LAMP test kit that detects porcine reproductive and respiratory syndrome virus as claimed in claim 1 is characterized in that described extraction RNA reagent LBB II-RNA contains following solution composition:
RA: get 1%2-mercaptoethanol 0.6ml, pH 8.01% Tris-HCL 0.6ml, 10mmol/ml EDTA 1ml sterilization distilled water 5.0ml;
RB:6mmol/ml guanidinium isothiocyanate 35ml;
RC: dehydrated alcohol 15ml;
RD：100u/μl?DNAse?A?20μl、10ml?DEPC?H
2O。
3. a kind of detection porcine reproductive and respiratory syndrome virus RT-LAMP test kit as claimed in claim 1 is characterized in that described reaction reagent contains following solution:
1) primer mixed solution PM:100pmol/ μ l F3 2.5 μ l, 100pmol/ μ l B3 2.5 μ l, 100pmol/ μ l FIP20 μ l, 100pmol/ μ l BIP 20 μ l, deionized water 5 μ l, total system 50 μ l;
2) reaction buffer mixture RM; Get 4mmol/ μ l sal epsom and get 50 μ l, 1.6mol/ μ l trimethyl-glycine 50 μ l, 10mmol/ μ ldNTPs 50 μ l, be dissolved in the 475 μ l PCR level water fixed molten to 625 μ l.Each reaction need be got 12.5 μ l RB.
3) reaction enzymes mixed solution EM; Get 16U/ μ l Bst large fragment DNA polysaccharase 24 μ l, 400U/ μ l AMV ThermoScript II 24 μ l, 1 μ mol/ μ l DTT, 2 μ l.Each reaction needed is got 1 μ l EB.
4) developer: developer constitutes (available from Invitrogen company) by single agents SYBRgreen I fluorescein, gets 1 μ lSYBRgreenI and is dissolved in 49 μ l PCR level water.Each reaction adds 1 μ l.
5) restriction enzyme SpuI:SpuI 1000u/ml (available from NEB company).
4. as claim 1,3 described a kind of RT-LAMP test kits that are used to detect porcine reproductive and respiratory syndrome virus is characterized in that wherein reacting the employed primer of primer mixed solution (PM) and are:
F3：CAGATCCGATTGCGGCAG
B3：TTCTACGCGGTGCAGGAA
FIP：CATCGGCTCGGATGGTGTCGATGGGCGACAATGTCCCTA
BIP：ACCCATGAGTGAGCCCGTACTCGACCCACTCAAAGGTTTCA
5. detection method that detects the RT-LAMP test kit of porcine reproductive and respiratory syndrome virus is characterized in that may further comprise the steps:
1) nucleic acid in the extraction testing sample;
2) preparation LAMP reaction solution;
3) get 5.0 μ l 1) described nucleic acid 5.5 μ l add step 2) in the reaction solution of preparation, reaction volume is 20 μ l the beginning eventually, the instantaneous centrifugal 30s of 10000rpm is with the mixing reaction solution;
4) put
℃ constant temperature 45min increases;
5) judged result is declared evaluation criteria and is: occurs greenly having porcine reproductive and respiratory syndrome virus in the testing sample; Occurring orange-yellow is not have porcine reproductive and respiratory syndrome virus in the testing sample.
6) take out adding SpuI enzyme system 9 μ L among the 1 μ L in the positive products of PRRSV-RT-LAMP reaction, totally 10 μ L systems are in 37 ℃ of warm bath effect 1h.
7) sepharose of preparation 1%.
8) with 6) in enzyme cut product 7) described in gel in carry out electrophoresis.Use Marker 2000 plus as the electrophoresis reference standard.
9) observations, declare evaluation criteria and be:
Lane 1 is that porcine reproductive and respiratory syndrome virus american type NSP2 variant (high-pathogenicity blue ear disease strain) RT-LAMP product is through SpuI enzyme effect rear electrophoresis result shown in the appearance accompanying drawing 8; Lane2 shown in the accompanying drawing 8 occurring is that the classical strain RT-LAMP of american type product is through SpuI enzyme effect rear electrophoresis result.
6. detection method that detects the RT-LAMP test kit of porcine reproductive and respiratory syndrome virus is characterized in that extracting sample RNA with LBB II-RNA may further comprise the steps:
Get 100 μ l cell cultures or serum sample or organize ground sample, behind multigelation 3 times, add sample equal-volume RA in the sample, vortex 15s, the centrifugal 1min of 12000g gets supernatant and places new pipe; The RB solution that in supernatant liquor, adds 3 times, vortex or concuss 90s leave standstill 3min; The RC solution that adds 1/3 volume, vortex 1min, the centrifugal 1min of 12000g, sample drying behind the abandoning supernatant placement 5min adds 10 μ l RD again, and dissolution precipitation is sample RNA.
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| CN102277445A (en) * | 2010-12-10 | 2011-12-14 | 中华人民共和国珠海出入境检验检疫局 | PRRS RT-LAMP detection method and detection kit |
| CN102621319A (en) * | 2012-03-16 | 2012-08-01 | 吉林大学 | Colloidal gold quick diagnosis test paper for distinguishing classic porcine reproductive and respiratory syndrome (PRRS) from highly pathogenic PRRS (HPRRS) |
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| CN106367529A (en) * | 2016-08-30 | 2017-02-01 | 中国农业科学院兰州兽医研究所 | RT-LAMP kit for detecting American type highly-pathogenic porcine reproductive and respiratory syndrome by adopting rapid developing one-step method |
| CN107502679A (en) * | 2017-09-21 | 2017-12-22 | 南京农业大学 | Differentiate LAMP primer and discrimination method and the application of PRRSV strain gene hypotypes |
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| CN108060211A (en) * | 2018-01-23 | 2018-05-22 | 中国热带农业科学院环境与植物保护研究所 | A kind of method for quantitatively detecting mango anthrax-bacilus |
| CN108060211B (en) * | 2018-01-23 | 2018-11-13 | 中国热带农业科学院环境与植物保护研究所 | A method of quantitatively detecting mango anthrax-bacilus |
| CN109750121A (en) * | 2018-12-29 | 2019-05-14 | 博奥生物集团有限公司 | Primer combination and its application in the testing product for preparing the high pathogenic strain of pig blue-ear disease poison american type |
| CN109880933A (en) * | 2019-02-19 | 2019-06-14 | 北京市动物疫病预防控制中心 | A kind of fluorescent visual quickly detects the RT-LAMP kit of porcine reproductive and respiratory syndrome american type strain |
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