CN108342513A - A kind of universal RT-PCR detection primers of different genotype grass carp reovirus and application - Google Patents
A kind of universal RT-PCR detection primers of different genotype grass carp reovirus and application Download PDFInfo
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Abstract
The invention discloses a kind of universal RT PCR detection primers of different genotype grass carp reovirus and applications.Pair of primers is used only in the method for the present invention, a PCR reaction can detect three kinds of genotype grass carp reovirus simultaneously（I type, II type and III type）, greatly improve detection efficiency, saved testing cost；The advantages that this method also has both high sensitivity, high specificity simultaneously, can play a significant role in the quick diagnosis of hemorrhagic disease of grass carp clinical sample and epidemiological survey.
Description
Technical field
The invention belongs to fishes virus molecular Biological Detection fields, and in particular to a kind of different genotype grass carp exhales intestines lonely
The universal RT-PCR detection primers of virus and application.
Background technology
Grass carp reovirus (grass carp reovirus, GCRV) is first plant of fishes virus of isolated in China.It should
Virion diameter is about 60~70nm, is in icosahedral symmetry, has double capsid, no cyst membrane, genome is by 11 merogenesis
The double-stranded RNA composition of section.The hemorrhagic disease of grass carp caused by grass carp reovirus supports China fresh water master the harm of kind grass carp
Greatly, the sound development of China's grass carp aquaculture is seriously affected since economic loss caused by the disease is up to ten several hundred million yuan every year.Mesh
The GCRV separation strains that preceding China has reported have more than 30, can be divided into 3 genotype according to gene order difference：GCRV I, II and III
Type, representative strains are respectively GCRV-873, GCRV-HZ08 and HGDRV (original name GCRV104).
The detection method that a variety of GCRV have had been established both at home and abroad, in addition to traditional cell separation, Electronic Speculum observation and virus
Outside genomic nucleic acids SDS-PAGE, RT-PCR, quantitative fluorescent PCR, reverse transcription loop-mediated isothermal amplification, dsRNA surveys have been also set up
Sequence method, monoclonal antibody detection and Nucleic acid sequence amplification and enzyme-linked immunosorbent assay binding assay etc..More than
Detection method respectively has the advantage of oneself, but these detection methods are just for some segment of specific genotype GCRV separation strains
Specific primer is designed, so primary first-order equation can only detect a kind of GCRV of genotype.To covering mesh first three genotype
GCRV, needing to design multipair primer or multiple reactions could complete so that detection time extends, and testing cost increases.Especially
Had the reports of multiple and different place GCRV separation strains again successively in recent years, and some new separation strains in gene order with it is existing
There is larger differences for separation strains, and the possibility of larger missing inspection is inevitably present using existing detection method, so comprehensive
Existing GCRV separation strains sequence is analyzed, a kind of the universal of simple and quick, applicable different genotype GCRV separation strains is established
RT-PCR detection method just seems particularly necessary.
The present invention is directed to 13 plants of different genotype grass carp reovirus separation strains, designs a pair of degenerate primers, establishes
A kind of universal RT-PCR detection method that can detect three kinds of genotype GCRV simultaneously using pair of primers, a PCR reaction,
Detection time is greatly saved, reduces testing cost, and has the advantages that high specificity, high sensitivity.For grass carp hemorrhage
The quick diagnosis of disease and epidemiological survey provide technical support.
Invention content
It is an object of the present invention to provide a kind of universal RT-PCR detections of different genotype grass carp reovirus
Primer, the primer are：GCRV-dpF:5 '-TAYGTVACMSCCMGRGGWGG -3 ' and GCRV-dpR:5’–
AADTGYTGYACCATGDYCTGC–3’。
It is another object of the present invention to provide a kind of universal RT-PCR inspections of different genotype grass carp reovirus
Survey the application of primer.
To achieve the goals above, the technical solution adopted in the present invention is：
A kind of universal RT-PCR detection primers of different genotype grass carp reovirus：
GCRV-dpF:5’–TAYGTVACMSCCMGRGGWGG–3’；
GCRV-dpR:5’–AADTGYTGYACCATGDYCTGC–3’.
A kind of universal RT-PCR detection primers of different genotype grass carp reovirus are preparing grass carp reovirus
Application in detection kit；
In above-described application, it is preferred that the kit includes：Degenerate primer (GCRV-dpF:5’–
TAYGTVACMSCCMGRGGWGG -3 ' and GCRV-dpR:5 '-AADTGYTGYACCATGDYCTGC -3 '), Ex Taq polymerases,
Ex Taq Buffer, dNTP mixtures.
Compared with prior art, the present invention has the following advantages：
The different separation strains of three kinds of genotype of degenerate primer pair (I, II, III type) GCRV used all have good degeneracy
Property, amplifiable different genotype GCRV separation strains all at present；
Pair of primers is used only in the detection kit of invention, a PCR reaction can detect three kinds of genotype grass simultaneously
Fish reovirus greatly improves detection efficiency, reduces testing cost；
The advantages that detection method also has both high sensitivity, high specificity simultaneously, can be in hemorrhagic disease of grass carp clinical sample
It plays a significant role in quick diagnosis and epidemiological survey.
Description of the drawings
Fig. 1 is 1% agarose gel electrophoresis figure of the specific detection PCR product of primer provided by the invention：
1~5 swimming lane in figure:Respectively using the nucleic acid of GSIV, KHV, CyHV-2, SVCV and ISKNV or reverse transcription product as mould
Plate；6~11 swimming lanes:Respectively with the anti-of GCRV-GZ1208, GCRV106, GCRV918, GCRV-HeNan988, HGDRV and CCRV
Transcription product cDNA is template；12 swimming lanes：Not plus the negative control of template.
Fig. 2 is 1% agarose gel electrophoresis figure of the sensitivity technique PCR product of primer provided by the invention：
1~9 swimming lane in figure：The different genotype GCRV plasmid copy numbers of gradient dilution are respectively 109~101copies/μ
L；10：Not plus the negative control of template.
Fig. 3 is 1% agarose gel electrophoresis figure of clinical sample testing result of primer provided by the invention：
1~15 swimming lane in figure:15 parts of hemorrhagic disease of grass carp samples to be acquired from Hubei, Jiangsu, Zhejiang, Guangdong and other places respectively
RNA reverse transcription products cDNA is template；16 swimming lanes：Not plus the negative control of template.
Specific implementation mode
With reference to embodiment, the present invention is further illustrated, but should not regard limitation of the present invention.This hair
The bright experimental method is if not otherwise specified the conventional scheme of this field；The reagent or material, if not otherwise specified,
Derive from commercial channel.
Embodiment 1：
The acquisition of degenerate primer：
For 13 plants of GCRV separation strains, I 2 plants of type, 10 plants of II type, 1 plant of III type design degenerate primer：
GCRV-dpF:5’–TAYGTVACMSCCMGRGGWGG–3’；
GCRV-dpR:5’–AADTGYTGYACCATGDYCTGC–3’；
In 590bp or so, wherein I types of GCRV are the clip size that above-mentioned primer expands in I, II, III types of GCRV
II type of 590bp, GCRV is 590bp or III type of 593bp, GCRV is 587bp.
Specific strain is as follows：GCRV-873 (I type, AF284502.1), GCRV-GZ1208 (I type, KU240075.1),
GCRV-HZ08 (II type, GQ896335.1), GCRV-GD108 (II type, HQ231199.1), GCRV106 (II type,
KC201167.1), GCRV918 (II type, KC201178.1), GCRV-JX02 (II type, KM880066.1), GCRV-HuNan794
(II type, KC238677.1), GCRV-Huan1307 (II type, KU254567.1), GCRV-AH528 (II type, KR180369.1),
GCRV-HeNan988 (II type, KC847321.1), GCReV109 (II type, KF712476.1), GCRV104 (III type,
JN967630.1)。
Embodiment 2：
A kind of universal RT-PCR detection method of different genotype grass carp reovirus, includes the following steps：
1) viral nucleic acid extracts：The cell for collecting virus infection, at -80 DEG C and multigelation 3 times under room temperature, 4
000r/min centrifuges 30min (Sigma, 3K-15), and supernatant is transferred in 35mL ultracentrifugation pipes, and 20 000r/min centrifuge 2h
(Optima L-80XP, Beckman-Coulter), suspension viral pellet is spare.RNA virus and DNA virus nucleic acid are used respectively
Trizol the and DNAzol reagents of Invitrogen companies extract, and concrete operations are carried out by reagent specification.The disease of extraction
Malicious RNA and DNA is stored in -80 DEG C respectively and -20 DEG C of refrigerators are spare.
The doubtful sample nucleic of the hemorrhagic disease of grass carp of acquisition is extracted, collects the viscera tissues such as the liver,spleen,kidney of sick fish in nothing
It in bacterium culture dish, is shredded with Sterile ophthalmic, the DPBS (Sigma) that 10 times of volumes (V/W) are added is transferred in glass homogenizer simultaneously
It is ground into tissue homogenate under ice bath, other steps are same as above.
2) it expands
Reverse transcription：Use PrimeScriptTMII 1st Strand cDNA Synthesis Kit reverse transcription reagent box
(TaKaRa, Dalian) synthesizes cDNA templates, and reverse transcription primer is Random 6mers (50 μM) 1 μ L, 8 μ L of viral RNA, totality
It is 20 μ L, concrete operations by specification carries out.
Pcr amplification reaction (is carried out) using detection kit：PCR reaction related reagents are all from Dalian TaKaRa companies,
50 μ L of total system, including：5 μ L reverse transcription products, 5 μ L 10 × Ex Taq Buffer (Mg2+Plus) (20mM), 4 μ L dNTP
Mixture (each 2.5mM), upstream and downstream degenerate primer (50 μM) each 1 μ L, 0.25 μ L TaKaRa Ex Taq (5U/ μ L), with super
Pure water polishing is to 50 μ L.
PCR response procedures：95 DEG C of 5min pre-degenerations, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 cycles, 72 DEG C of extensions
10min, 4 DEG C of heat preservations.
The judgement of testing result：5 μ L pcr amplification products are taken, is detected with 1.0% agarose gel electrophoresis, is placed in gel
It is imaged in imaging system, if electrophoresis picture, which is shown at 590bp sizes, band, result is the positive；Such as without any band, then
As a result it is feminine gender；Wherein I types of GCRV are that II type of 590bp, GCRV is 590bp or III type of 593bp, GCRV is 587bp.
Embodiment 3：
The detection of primer specificity：
Use the method detection channel catfish reovirus (CCRV, with I type very high homology), big described in embodiment 2
Salamander irido virus (GSIV), Koi herpesvirus (KHV), carp herpesvirusⅡtype (CyHV-2), huichun viremia virus
(SVCV) and infectious spleen and kidney necrosis virus (ISKNV) and above-mentioned 13 kinds of grass carp reovirus., 5 μ L PCR amplifications is taken to produce
Object is detected with 1.0% agarose gel electrophoresis.
Electrophoresis detection the result shows that, with three kinds of genotype grass carp reovirus and channel catfish reovirus
(CCRV) cDNA is that the 590bp that template can expand to obtain expected size or so purpose band, sequencing result show in fact
Border length is consistent with described in embodiment 2；And with other fish common virus nucleic acid such as GSIV, KHV, CyHV-2, SVCV and ISKNV
Or reverse transcription product cannot amplify any band for template (partial results are as shown in Figure 1).It should be noted that CCRV its
It is exactly the GCRV for the I types for infecting Channel-catfish fishes in fact, through sequencing, which is also 590bp.
Embodiment 4：
The detection of primer sensitivity：
The recombinant plasmid of GCRV-GZ1208 (I type), GCRV106 (II type) and HGDRV (III type) are carried out 10 with ultra-pure water
Times gradient serial dilution, makes its concentration be followed successively by 109Copy/L~10 μ1Copy/μ L are made with the plasmid as template of gradient dilution
PCR amplification is carried out with degenerate primer, detects the sensitivity of this method.
Sensitivity technique result：When plasmid is diluted to 102When copy/μ L, can still it expand in three kinds of genotype GCRV templates
Increase and specific purpose band (Fig. 2).I.e. I type of this method pair, II type, the minimum detection limit of III type GCRV nucleic acid be respectively 380,
250 and 380 copy numbers.
Embodiment 5：
The detection result of primer pair clinical sample：
Clinical detection sample be this laboratory in 2015~2017 years from the areas grass carps Zhu Yang such as Hubei, Jiangsu, Zhejiang, Guangdong
The 15 parts of hemorrhagic disease of grass carp samples for acquiring and preserving.
PCR product electrophoresis result is shown, other than negative control, 15 parts of clinical samples have expanded the spy of 590bp or so
Anisotropic band, so 15 parts of samples of detection are all positive (Fig. 3).
Sequence table
<110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120>A kind of universal RT-PCR detection primers of different genotype grass carp reovirus and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
taygtvacms ccmgrggwgg 20
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
aadtgytgya ccatgdyctg c 21
Claims (2)
1. a kind of universal RT-PCR detection primers of different genotype grass carp reovirus：
GCRV-dpF: 5’ –TAYGTVACMSCCMGRGGWGG – 3’;
GCRV-dpR: 5’ –AADTGYTGYACCATGDYCTGC – 3’.
2. application of the primer described in claim 1 in preparing grass carp reovirus detection kit.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876809A (en) * | 2012-10-18 | 2013-01-16 | 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 |
| CN103484568A (en) * | 2013-09-18 | 2014-01-01 | 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 |
| CN103539842A (en) * | 2013-10-25 | 2014-01-29 | 中国水产科学研究院珠江水产研究所 | Recombinant protein coded by grass carp reovirus (GCRV) type-II S10 gene, polyclonal antibody prepared from recombinant protein and application of recombinant protein |
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- 2018-05-10 CN CN201810443444.0A patent/CN108342513B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876809A (en) * | 2012-10-18 | 2013-01-16 | 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 |
| CN103484568A (en) * | 2013-09-18 | 2014-01-01 | 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 |
| CN103539842A (en) * | 2013-10-25 | 2014-01-29 | 中国水产科学研究院珠江水产研究所 | Recombinant protein coded by grass carp reovirus (GCRV) type-II S10 gene, polyclonal antibody prepared from recombinant protein and application of recombinant protein |
Non-Patent Citations (2)
| Title |
|---|
| YUDING FAN ET.AL: "Identification and genomic characterization of a novel fish reovirus, Hubei grass carp disease reovirus, isolated in 2009 in China", 《J GEN VIROL.》 * |
| 张爱联主编: "《生物化学与分子生物学实验教程》", 31 January 2009 * |
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