CN102534050B - Triple polymerase chain reaction (PCR) detecting primer group, reagent kit and method for diagnosing grass carp reovirus - Google Patents
Triple polymerase chain reaction (PCR) detecting primer group, reagent kit and method for diagnosing grass carp reovirus Download PDFInfo
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Abstract
The invention discloses a triple polymerase chain reaction (PCR) detecting primer group, a reagent kit and a method for diagnosing grass carp reovirus. The method disclosed by the invention is a triple RR-PCR detecting method capable of simultaneously detecting three kinds of subtypes of the grass carp reovirus: HZ08 strain types, 104 strain types and JX09-01 strain types, the subtypes of the grass carp reovirus can be identified when the grass carp reovirus is detected, the valuable time is obtained for the providing of prevention and control measures in a targeted way and more precise treatment according to diseases, and the important significance is realized on the effective control of grass carp bleeding diseases. When being applied to the detection of the grass carp reovirus, the primer group and the reagent kit disclosed by the invention have the advantages of accuracy, flexibility, high speed, cost saving and the like.
Description
Technical field
The invention belongs to the viral molecular biology detection field, the triple PCR that is specifically related to the GCRV different subtype detects, and comprises primer sets, test kit and the method for detecting.
Background technology
GCRV (Grass carp reovirus, GCRV) is subordinate to Aquareovirus, is Reoviridae one newcomer, is the first strain fishes virus that separate the China's Mainland.This virus causes that mainly the grass carp kind in Asian countries's freshwater aquicultures such as China, Vietnam, Burma in the fingerling stage, hemorrhagic disease occurs, and mortality ratio can be up to more than 60%.This viral prevalence is wide, harm is large, mortality ratio is high, morbidity season is long, the serious threat fish production.GCRV tool double capsid, virus particle mean diameter are 60 nm-70nm, and icosahedron is symmetrical, and without cyst membrane, genome is comprised of 11 segmented double-stranded RNAs.Oneself is through having reported nearly 20 strain isolateds at present, comprise GCRV854, GCRV861, GCRV873, GCRV875, GCRV876, GCRV991, GCRV H962, ZV-8802, GCRV-854, GCRV HZ08, GCRV JX09-01, GCRV JX09-02, GCRV GD10, GCRV GCRV104 strain etc., different isolates is at genome sequence, genome banding pattern, cytopathy, differ greatly to the aspects such as virulence of grass carp.Up to the present, have only have GCRV 873 strains, GCRV HZ08, the GCRV GD10 of report to complete complete genome sequencing, and other strain has only been completed the examining order of part sections or partial sequence.The GCRV more complicated, each gene segment of different isolates exists reprovision and antigenic drift phenomenon, makes at present and also on serology or genotype, it is not carried out Subtypes.But by existing strain isolated sequence information, at least three classes should be arranged, all kinds of representative strains are respectively: the first kind is 873 strains and JX09-01 strain, and Equations of The Second Kind is HZ08 strain and GD10 strain, and the 3rd class is 104 strains.Have in relevant research report and mention the discussion of it being carried out gene type, namely respectively one, two and three classes are divided into I, II and III type according to gene order difference.(1, Ceng Weiwei, Wang Qing, Liu Yongkui, etc. the separation of a strain GCRV low virulent strain, evaluation and immunogenicity initial analysis.The hydrobiont journal, 2011.35 (5): 790-795; 2, Liu Baoqin, Ceng Weiwei, Wang Qing, etc. foundation and the Preliminary Applications of GCRV HZ08 strain FQ-PCR detection method).The strain of same hypotype, its corresponding each sections DNA homolog is all more than 95%.Current, in the epidemic strain that all parts of the country are separated to, three class hypotypes all have report, and the independent infection that has also has polyinfection.Epidemic characteristic and GCRV molecular biological characteristic according to hemorrhagic disease of grass carp, between strains different from same hypotype, total conserved sequence designs respectively primer, foundation all can diagnose simultaneously for present known all popular GCRVs, and can identify that triple RT-PCR of its hypotype are quick, the specific detection technology.
Virus separation, electron microscopic observation, nucleic acid belt type analysis are still the method that GCRV generally adopts that detects so far, but these method operation more complicated can not be carried out quick diagnosis to virus, are unfavorable for the timely and effective anti-system of hemorrhagic disease of grass carp.Polymerase chain reaction (PCR) can enlarge millions of times to certain fragment gene within a few hours.This technology high specificity, susceptibility is high, detection is quick, has been widely used in each field of the subjects such as medical science, microbiology, veterinary science.Multiplex PCR is a kind of special PCR form, and its outstanding feature is that a PCR can detect several genes simultaneously, is particularly useful for the cause of disease complexity, and the more cause of disease of genotype detects.
Summary of the invention
One object of the present invention is to provide a kind of triple PCR of GCRV to detect primer sets
Another object of the present invention is to provide a kind of GCRV triple PCR detection kit that contains above-mentioned primer sets.
Another object of the present invention is to provide a kind of triple PCR detection method of GCRV.
The technical solution adopted in the present invention is:
The triple PCR of GCRV detects primer sets, is comprised of following 3 pairs of primers:
P01-F：5' GCC ACC TTT GAG CGC GAG AC 3'(SEQ ID NO：1)；
P01-R：5' GTT AGG GCG GAA AGC ATA CCA GA 3'(SEQ ID NO：2)；
P02-F：5' GCT GAT GCT GCA GAC GGC TAA AC 3'(SEQ ID NO：3)；
P02-R：5' TAA TTG CCT GCT GCG CTG ACT 3'(SEQ ID NO：4)；
P03-F：5' GGC GGC ATG AAT ATG TAT CGA CT 3'(SEQ ID NO：5)；
P03-R：5' TAT GTG ATT ACG CGG GTC AG 3' (SEQ ID NO：6)。
The triple PCR detection kit of GCRV comprises primer liquid, Tap archaeal dna polymerase, PCR buffer, dNTP mixture, and described primer liquid contains above-mentioned primer sets (SEQ ID NO1 ~ 6).
The triple PCR detection method of GCRV comprises the steps:
1) extract total tissue RNA to be checked;
2) treat the total RNA of sample product and carry out reverse transcription reaction;
3) add in the PCR reaction system after three pairs of primers in above-mentioned primer sets (SEQ ID NO:1 ~ 6) are mixed in the Isoequivalent weight ratio, take reverse transcription product cDNA as template, carry out pcr amplification reaction;
4) judge according to the pcr amplification result whether sample to be checked infects the genotype of GCRV and judgement virus.
Preferably, described PCR reaction system is 25 μ L, comprise primer sets claimed in claim 1, wherein the final concentration of every primer (SEQ ID NO:1 ~ 6) is 1pmol/ μ L, 2.5 μ L 10 * PCR buffer, 5U Tap archaeal dna polymerase, final concentration are respectively the dNTP mixture of 0.1mM, as template, water is mended to 25 μ L with 10 μ L reverse transcription product cDNA.
Preferably, the pcr amplification reaction program is: 95 ℃ of ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 53.6 ℃ of annealing 30s, 72 ℃ are extended 40s, circulate 30 times, and 72 ℃ are extended 10min.
Beneficial effect of the present invention is:
(1) the present invention has set up and can detect simultaneously three kinds of hypotypes of GCRV: triple RR-PCR detection methods of HZ08 plant type, 104 plant types, JX09-01 plant type, can be when detecting GCRV, identify its hypotype, for proposing targetedly prevention and control measure and the scheme that due to illness treats has more accurately been striven for the quality time, effective control of hemorrhagic disease of grass carp is significant;
(2) use the detection that primer sets of the present invention and test kit carry out GCRV have accurately, sensitive, fast, the advantage such as saving cost.
Description of drawings
Fig. 1 is the 1% agarose gel electrophoresis figure (1: the negative control that does not add template as a result that uses the inventive method amplification GCRV products therefrom, 2: take GCRV HZ08 strain RNA reverse transcription product cDNA as template, 3: only GCRV 108 strain RNA reverse transcription product cDNA are template, 4: take GCRV JX09-01 strain RNA reverse transcription product cDNA as template, 5: take GCRV HZ08 strain and 108 strain RNA reverse transcription product cDNA as template, 6: take GCRV HZ08 strain and JX09-01 strain RNA reverse transcription product cDNA as template, 7: take GCRV 104 strains and JX09-01 strain RNA reverse transcription product cDNA as template, 8: with GCRV HZ08 strain, 104 strains and JX09-01 strain RNA reverse transcription product cDNA are template, M:DL2000 Marker),
Fig. 2 is the 1% agarose gel electrophoresis figure (1: the negative control that does not add template as a result of the specificity experiment PCR product of the inventive method; 2-5: respectively take the nucleic acid of KHV, IHNV, LMBV and ISKNV as template; 6-8: respectively take GCRV HZ08 strain, GCRV JX09-02 strain and GCRV JX10-01 strain RNA reverse transcription product cDNA as template; 9:GCRV 104 strain RNA reverse transcription product cDNA are template; 10-12: the GCRV RNA reverse transcription product cDNA that is separated to from Guangxi take GCRV JX09-01 strain, GSRV strain and a strain respectively is as template; 13-14: respectively to be separated to the GCRV RNA reverse transcription product cDNA of polyinfection as template from Hubei and Jiangxi; M:DL2000 Marker);
Fig. 3 is the 1% agarose gel electrophoresis figure (1: the negative control that does not add template as a result of the sensitivity experiments PCR product of the inventive method; 2-8: be respectively 5 * 10 with GCRV HZ08 strain RNA reverse transcription product cDNA concentration
5～5 * 10
-1Copy/μ l is template; 9-15: be respectively 5 * 10 with GCRV 108 strain RNA reverse transcription product cDNA concentration
5～5 * 10
-1Copy/μ l is template; 16-22: be respectively 5 * 10 with GCRV JX09-01 strain RNA reverse transcription product cDNA concentration
5～5 * 10
-1Copy/μ l is template; M:DL2000 Marker);
Fig. 4 is the clinical sample detected result (1: the negative control that does not add template of the inventive method; 2-19: respectively take from Jiangxi, 18 parts of hemorrhagic disease of grass carp sample RNA reverse transcription product cDNA gathering of Hubei, Guangxi, Guangdong, Zhejiang, Shandong, Hunan are as template; M:DL2000 Marker).
Embodiment
The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.
1. test with strain and pathological material of disease collection
Koi herpesvirus 3 types (KHV), infectious hematopoietic necrosis's poison (IHNV), Micropterus salmoides irido virus (LMBV), infectious spleen and kidney necrosis virus (ISKNV), GCRV HZ08 strain, GCRV JX09-01, GCRV JX10-01 strain, GCRV JX09-02, GCRV 104 strains are separated by this experiment and preserve, and GSRV is available from ATCC.The bacterial isolate pathological material of disease is that the fish body viscera tissue of suffering from corresponding fish disease gathers gained, and pathological material of disease tissue is stored in after by aseptic collection and contains in two phosphate buffered saline buffers that resist, and every pipe contains 25～30ml damping fluid ,-80 ℃ of preservations.
The design of primer and synthetic
The all gene orders of existing GCRV in GENBANK have been analyzed, chosen one section 532bp conserved sequence of JX09-01 and 873 strain S6 genes, the conserved sequence of one section 196bp of HZ08 and GD10 strain S6 gene, the sequence of one section 297bp of 104 strain S8 genes is as the amplification target, and three pairs of primers have been synthesized in design:
P01-F: 5' GCC ACC TTT GAG CGC GAG AC 3'(SEQ ID NO：1)；
P01-R:5' GTT AGG GCG GAA AGC ATA CCA GA 3'(SEQ ID NO：2)；
P02-F 5' GCT GAT GCT GCA GAC GGC TAA AC 3'(SEQ ID NO：3)；
P02-R: 5' TAA TTG CCT GCT GCG CTG ACT 3'(SEQ ID NO：4)；
P03-F:5' GGC GGC ATG AAT ATG TAT CGA CT 3'(SEQ ID NO：5)；
P03-R: 5' TAT GTG ATT ACG CGG GTC AG 3' (SEQ ID NO：6)；
The purpose expanding fragment length is respectively 532bp, 297bp and 196bp.
Detection kit forms
Primer liquid: obtain after each in above-mentioned primer sets mixed by Isoequivalent weight concentration primer (SEQ ID NO:1 ~ 6), concentration is respectively 25pmol/ μ L;
The Tap archaeal dna polymerase, 5 U/ μ L;
10 * PCR buffer consists of 500mM KCl, 100mM Tris-HCl (pH9.0), 1% Triton X-100,20mM MgCl
2
DNTP mixture, concentration are 2.5 mM.
The extraction of viral RNA
Press the Trizol test kit (available from Invitrogen company, article No.: specification sheets 15596-026) or extract test kit (available from Qiagen company according to Qiagen RNA, article No.: specification sheets step 74104), extract RNA from the fish body viscera tissue that infects corresponding virus, as the template of RT-PCR reaction.The key step that the Trizol method is extracted total RNA is as follows: get tissue sample and add 500 μ L Trizol reagent, after mixing standing 5 minutes, add 400 μ L chloroforms, vibration mixing 1min, 12000rpm gets supernatant and adds the equal-volume Virahol in 4 ℃ of centrifugal 15min, puts upside down mixing, place 15min for 4 ℃, 12000rpm abandons supernatant in 4 ℃ of centrifugal 15min, washes precipitation with 70% ethanol, 12000rpm is in 4 ℃ of centrifugal 5min, with 30 μ L without the resuspended precipitation of RNAse water.
Amplification
Reverse transcription: the reverse transcription primer uses P01, P02 and each 2pmol of P03 primer, and viral RNA 1 μ L uses the AMV reverse transcription test kit (Cat.Nos.DRR019A) of TAKARA company, totally 10 μ L systems, process is carried out with reference to specification sheets.
Pcr amplification reaction (utilize 3 detection kit react): use 25 μ L reaction systems, comprise: 10 μ L reverse transcription products, 1 μ L primer liquid (final concentration of primer SEQ ID NO:1 ~ 6 is 1pmol/ μ L), 5U LA Taq enzyme, 2.5 μ L 10 * PCR reaction buffer, 1 μ L dNTP mixture (final concentration is 0.1mM), water polishing to 25 μ L.
The pcr amplification reaction program: 95 ℃ of 5min denaturations, 94 ℃ of 30s, 53.6 ℃ of 40s, 72 ℃ of 40s, 30 circulations, 72 ℃ are extended 10min.Pcr amplification product is identified with 1% agarose gel electrophoresis.
The detected result judging criterion: if only amplified the product of 196bp from sample to be checked, this sample is GCRV HZ08 plant type, i.e. II type; If only amplified the product of 297bp from sample to be checked, this sample is GCRV 104 plant types, i.e. III type; If only amplified the product of 536bp from sample to be checked, this sample is GCRV JX09-01 plant type, i.e. I type; If amplify simultaneously the two or three in 196bp, 297bp and 536bp, this sample is amphitypy in this three C-type virus C or the common infection of three types; If do not amplify any fragment in 196bp, 297bp and 536bp from sample to be checked, this sample does not contain at present known GCRV infection.
This experimental result shows, the purpose fragment that obtains 196bp as template can increase take HZ08 strain hypotype GCRV; The purpose fragment that obtains 297bp as template can increase take 104 strain hypotype GCRV, the purpose fragment that can increase and obtain 532bp with JX09-01 strain hypotype GCRV template; Can increase as template with two or three GCRV in HZ08 strain, 104 strains and JX09-01 strain and obtain two or three purpose fragment in 196bp, 297bp and 532bp.Take other aquatic viruses as template, can not increase obtains any fragment (see figure 1).
Specific test
RT-PCR amplification method according to 5 detects respectively KHV, IHNV, LMBV, ISKNV, GCRV HZ08 strain, GCRV JX09-02, GCRV JX10-01 strain, GCRV JX09-01 strain, GSRV strain and separation and detection and arrives but unnamed GCRV, and pcr amplification product is identified with 1% agarose gel electrophoresis.
Electrophoresis result shows: the GCRV nucleic acid take different subtype all can be expanded the purpose fragment that obtains corresponding size as template, and obtains any fragment (see figure 2) as template can not increase take other fish common virus nucleic acid.
Sensitivity test
Get each strain of GCRV (HZ08 plant type, 104 plant types, the JX09-01 plant type) each 100 μ l of enchylema, use RNA to extract test kit ((available from Qiagen company, article No.: 74104) extract RNA, the RNA concentration of using the fluorescent quantitation method to measure is 5 * 10
5Copy/μ l, after 10 times of serial dilutions, 1 μ l template is used in each RT-PCR reaction (method is with 5), and pcr amplification product is identified with 1% agarose gel electrophoresis.
Electrophoresis result shows: the inventive method can detect the virus quantity (seeing Fig. 3) that the nucleic acid minimum quantity is about 50 copies.
The clinical application test
Use present method to identify to 18 parts of hemorrhagic disease of grass carp samples, 14 parts of equal can amplifications of sample that are separated to GCRV obtain the purpose fragment, and 4 parts of samples that are not separated to GCRV can not obtain any fragment, and coincidence rate is that 100%(sees Fig. 4 as a result).
Above embodiment does not consist of any restriction to scope of the present invention only for introducing preferred case of the present invention.To those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention all should be regarded as a part of the present invention.
＜110〉China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
＜120〉triple PCR that is used for the diagnosis GCRV detects primer sets, test kit and method
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<170> PatentIn version 3.5
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Claims (2)
1. the triple PCR of GCRV detects primer sets, is comprised of following 3 pairs of primers:
Primer pair 1
P01-F：5' GCC ACC TTT GAG CGC GAG AC 3'(SEQ ID NO：1)；
P01-R：5' GTT AGG GCG GAA AGC ATA CCA GA 3'(SEQ ID NO：2)；
Primer pair 2
P02-F：5' GCT GAT GCT GCA GAC GGC TAA AC 3'(SEQ ID NO：3)；
P02-R：5' TAA TTG CCT GCT GCG CTG ACT 3'(SEQ ID NO：4)；
Primer pair 3
P03-F：5' GGC GGC ATG AAT ATG TAT CGA CT 3'(SEQ ID NO：5)；
P03-R：5' TAT GTG ATT ACG CGG GTC AG 3' (SEQ ID NO：6)。
2. the triple PCR detection kit of GCRV, comprise primer liquid, Tap archaeal dna polymerase, PCR buffer, dNTP mixture, and described primer liquid contains primer sets claimed in claim 1.
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| CN102876809B (en) * | 2012-10-18 | 2014-05-14 | 中国水产科学研究院长江水产研究所 | GCRV (grass carp reovirus) RT-PCR (reverse transcription-polymerase chain reaction) detection reagent kit and detection method |
| CN103484568B (en) * | 2013-09-18 | 2015-06-24 | 中国水产科学研究院珠江水产研究所 | Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II |
| CN104388588B (en) * | 2014-11-11 | 2017-02-15 | 中国水产科学研究院珠江水产研究所 | NASBA-ELISA (nucleic acid sequence-based amplification-enzyme-linked immuno sorbent assay) detection primer, probe and kit for type II grass carp reovirus |
| CN105176980B (en) * | 2015-09-05 | 2016-05-18 | 中国水产科学研究院黄海水产研究所 | A kind of multiple PCR method that can synchronously detect 7 kinds of fishes virus |
| CN106811550B (en) * | 2017-03-11 | 2020-09-18 | 中国水产科学研究院珠江水产研究所 | Grass carp reovirus type II vaccine strain and wild strain identification primer, kit containing grass carp reovirus type II vaccine strain and wild strain identification primer and diagnostic detection method |
| CN108504783A (en) * | 2018-06-07 | 2018-09-07 | 安徽省农业科学院水产研究所 | A kind of detection method of grass carp reovirus |
| CN110592269A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) |
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