CN102876811A - RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus) - Google Patents

RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus) Download PDF

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Publication number
CN102876811A
CN102876811A CN2012104030539A CN201210403053A CN102876811A CN 102876811 A CN102876811 A CN 102876811A CN 2012104030539 A CN2012104030539 A CN 2012104030539A CN 201210403053 A CN201210403053 A CN 201210403053A CN 102876811 A CN102876811 A CN 102876811A
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svcv
lamp
reaction
detection kit
primer
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张辉
曾令兵
周勇
范玉顶
徐进
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Yangtze River Fisheries Research Institute CAFS
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Yangtze River Fisheries Research Institute CAFS
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Abstract

The invention discloses a RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and a RT-LAMP detection method for SVCV (spring viremia of carp virus). The RT-LAMP detection kit of SVCV comprises 10*ThermoPol Reaction buffer, Bst DNA (deoxyribonucleic acid) polymerase, dNTPs (deoxynucleoside triphosphates), AMV reverse transcriptase, outer primer F3 and B3, inner primer FIP, BIP and Betaine, MgC 12, DTT, and 1000*SYBR Green I. The RT-LAMP detection kit is simple, fast, and high in specificity and sensitivity, only needs a water bath kettle or a metal bath to accurately detect SVCV in samples in 2 hours, can detect SVCV of infected tissues of sick fish, can detect cells infected by SVCV, and is suitable for fast field detection of SVCV.

Description

A kind of SVCV RT-LAMP detection kit and detection method
Technical field
The invention belongs to fishes virus detection technique field, be specifically related to a kind of SVCV RT-LAMP detection kit, also relate to a kind of method of utilizing SVCV RT-LAMP detection kit to detect SVCV.
Background technology
SVCV (Spring viraemia of carp virus, SVCV), belong to Mononegavirales (Mononegavirales), Rhabdoviridae (Rhabdoviridae), vesicular stomatitis virus belongs to the tentative species of (Vesiculovirus), and a serotype is only arranged at present.The spring viremia that SVCV causes (Spring Viraemia of Carp, SVC) is a kind of acute, hemorrhagic the prevailing disease that hyperinfection is arranged, and the serious harm fish production also can cause crushing blow.OIE (OIE) classifies it as must report important epidemic disease.Classifying it as class animal epidemic in the new animal epidemic registers of promulgating of China in 2008, has been that first is also that a unique fishes virus epidemic disease is listed in a class transmissible disease since founding of New.
SVC is huge to the aquaculture harm that particularly cyprinid fish cultivates.Therefore owing to temporarily not preventing and treating preferably medicine and method, to this, sick prevention and control need to rely on the diagnosis in laboratory, set up that detection method is most important fast and accurately." gold standard " that SVCV detects is by the cell cultures isolated viral, utilizes immunological technique or Protocols in Molecular Biology to be identified, this process length consuming time, program complexity.Prepare in addition the High-titre antiserum difficulty, also limited the use of immunological method.GB and rower that OIE hydrocoles medical diagnosis on disease handbook, China are detected about SVCV, classify RT-PCR as diagnostic method, but it is more complicated that the RT-PCR method operates, higher to instrument and personnel's requirement, be not suitable for field quick detection and basic unit's popularization and application.
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology is a kind of novel isothermal nucleic acid amplification method (International Patent Publication No. WO 00/28082) that Notomi equals report in 2000,4 LAMP primers of 6 site designs for target gene, utilize a kind of archaeal dna polymerase (Bst archaeal dna polymerase) with strand displacement activity, under constant temperature fast, efficient, high special, amplified target sequence with sensitivity, be suitable for the on-the-spot and better simply laboratory of experiment condition and fast detect.Reverse transcription loop-mediated constant-temperature amplification (RT-LAMP) is to add a certain amount of reversed transcriptive enzyme in reaction solution, realizes the step amplification of RNA.Introduce improving one's methods of a pair of Loop primer, further Reaction time shorten.
Summary of the invention
An object of the present invention is to be to provide a kind of SVCV RT-LAMP detection kit, the N gene fragment coding region sequence (U18101) of the SVCV strain of announcing according to GenBank, application PrimerExplorer V4(http: //primerexplorer.jp/elamp4.0.0/index.html) online software design Auele Specific Primer, utilize the specific region of RT-LAMP technology amplified target gene, from molecular level, SVCV is carried out to rapid detection, there are the characteristics of easy, quick, high specific and susceptibility.
Another object of the present invention is to be to provide a kind of method of utilizing SVCV RT-LAMP detection kit to detect SVCV, present method only needs a water-bath or the metal bath SVCV in can in 2 hours, accurately detecting sample, can detect the sick fish tissue that SVCV infects, can detect the cell (for example EPC cell) that SVCV infects, be highly suitable for the field quick detection of SVCV.
To achieve these goals, the present invention is by the following technical solutions:
A kind of SVCV RT-LAMP detection kit comprises following composition:
This test kit comprises following composition: 10 * ThermoPol Reaction Buffer; Bst archaeal dna polymerase (NEB); DNTPs; AMV reversed transcriptive enzyme (BBI); Outer primer F3 and B3, inner primer FIP and BIP, Betaine(Sigma), MgCl 2, DTT(Invitrogen), 1000 * SYBR Green I(Invitrogen).
F3:5,-GGGATAGCTTCGGACACAAG-3,;
B3:5,-CCATCAGCAAAGTCCGGTAT-3,;
FIP:5,-GTTTCCCATCTGGTAGCCCGTCTTTTAGATCGGGCCTTTTAACCTG-3,;
BIP:5,-AGGTGAGTGCTGAGGATGATGCTTTTTTGCCCTTCCCACTCTGT-3,。
A kind of method of utilizing SVCV RT-LAMP detection kit to detect SVCV, the steps include:
1, obtaining of viral RNA:
Adopt or lS reagent or post absorb-elute method etc. are extracted the total RNA of sample.
2, RT-LAMP amplification:
Adopt 25 μ l reaction systems, comprising: each 0.8 μ M of inner primer FIP and BIP, each 0.1 μ M of outer primer F3 and B3, dNTPs 1mM, Betaine 0.5M, DTT 4mM, MgCl 22-10mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l.Select after the time range of the temperature range of 55-65 ℃ and 30-120 minute carries out the RT-LAMP reaction 80 ℃ of deactivations 2 minutes.
3, detected result is judged, one of available following three kinds of methods are carried out the judgement of result:
While 1) amplified reaction occurring, the pyrophosphate ion of separating out from dNTPs and the magnesium ion reaction solution form white magnesium pyrophosphate precipitation, by naked eyes, judge: detector tube occurs obviously muddy positive, has no muddy negative.
2) reaction tubes of every 25 μ l systems adds 1000 * SYBR Green I(Invitrogen) 1-2 μ l, the 1-5min observations, it is green positive that reaction solution turns, and keeps orange negative.
3) get amplified production, use 2%(w/v) agarose gel electrophoresis after, be placed in the gel imaging system imaging, electrophoresis Image Display LAMP characteristic scalariform band, result is positive; As without any band, result is negative.
A kind of method (top condition) of utilizing SVCV RT-LAMP detection kit to detect SVCV, the steps include:
1, obtaining of viral RNA:
Adopt or lS reagent or post absorb-elute method etc. are extracted the total RNA of sample, and template ribonucleic acid is placed in rapidly ice bath after 5 minutes pre-treatment at 95 ℃ can increase the sensitivity of detection.
2, RT-LAMP amplification:
Adopt 25 μ l reaction systems, comprising: each 0.8 μ M of inner primer FIP and BIP, each 0.1 μ M of outer primer F3 and B3, dNTPs 1mM, Betaine 0.5M, DTT 4mM, MgCl 28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l.Reaction tubes after 64 ℃ of incubations carry out the RT-LAMP reaction in 60 minutes, 80 ℃ of deactivations 2 minutes.
3, detected result is judged, one of available following three kinds of methods are carried out the judgement of result:
While 1) amplified reaction occurring, the pyrophosphate ion of separating out from dNTPs and the magnesium ion reaction solution form white magnesium pyrophosphate precipitation, by naked eyes, judge: detector tube occurs obviously muddy positive, has no muddy negative.
2) reaction tubes of every 25 μ l systems adds 1000 * SYBR Green I(Invitrogen) 1-2 μ l, the 1-5min observations, it is green positive that reaction solution turns, and keeps orange negative.
3) get amplified production, use 2%(w/v, below identical) agarose gel electrophoresis after, be placed in the gel imaging system imaging, electrophoresis Image Display LAMP characteristic scalariform band, result is positive; As without any band, result is negative.
Compared with prior art, the present invention has the following advantages:
1, the invention discloses a kind of SVCV RT-LAMP detection kit;
2, specificity is good, can effectively detect SVCV virus;
3, rapidly and efficiently, approximately 1 hour detection time, be highly suitable for the field quick detection of SVCV;
4, do not need special reagent and equipment, testing cost is low;
5, identify simply, by pyrophosphate ion and the magnesium ion reaction solution of separating out from dNTPs, form white magnesium pyrophosphate precipitation, but through naked eyes judged result just, by adding 1000 * SYBR Green I, the sensitivity that can improve result.
The accompanying drawing explanation
The specific schematic diagram that Fig. 1 is a kind of SVCV RT-LAMP test kit detected result.
Swimming lane from left to right is followed successively by total RNA of the blank of water, normal EPC cell total rna, CRV infection CCO cell, total RNA, the SVCV of GCRV-104 strain infection CIK cell and infects total RNA, the DL2 of EPC cell, 000Marker.
Embodiment
Following example further illustrates the present invention, but should not regard limitation of the present invention.Unless otherwise noted, agents useful for same of the present invention is the production of Shanghai Sheng Gong bio-engineering corporation.
Embodiment 1:
A kind of SVCV RT-LAMP detection kit comprises following composition:
It comprises following composition this test kit: 10 * ThermoPol Reaction Buffer; Bst archaeal dna polymerase (NEB); DNTPs; AMV reversed transcriptive enzyme (BBI); Outer primer F3 and B3, inner primer FIP and BIP, Betaine(Sigma), MgCl 2, DTT(Invitrogen), 1000 * SYBR Green I(Invitrogen).
F3:5,-GGGATAGCTTCGGACACAAG-3,
B3:5,-CCATCAGCAAAGTCCGGTAT-3,
FIP:5,-GTTTCCCATCTGGTAGCCCGTCTTTTAGATCGGGCCTTTTAACCTG-3,
BIP:5,-AGGTGAGTGCTGAGGATGATGCTTTTTTGCCCTTCCCACTCTGT-3,。
Embodiment 2:
The optimization of the Mg2+ of SVCV RT-LAMP detection kit different concns
One, get sample extraction viral RNA to be checked:
Cultivate carp epithelium papilloma cell (EPC cell) to confluent monolayer, the sucking-off nutrient solution, the dosage that the infection multiplicity of take is 0.1, inoculation 1mL removes the SVCV (Zhang Peng of cell debris through the centrifugal 5min of 4000r/min, Liu's Polygonum, Chen Xiaoxuan, Deng. the preparation of SVCV monoclonal antibody and CHARACTERISTICS IDENTIFICATION thereof [J], China's Preventive Veterinary Medicine newspaper, 2011, 33(4): 305-308.) cell toxicant material, add the Polybrene(final concentration 10 μ g/ml of 10 μ L), be placed in 26 ℃ of incubators and adsorb 1h, make virus absorption onto cell, in adsorption process, every 20min rocks culturing bottle once gently, make that virus liquid and cell monolayer are full and uniform to be contacted, after absorption 1h, virus liquid is abandoned in suction, the nutrient solution that adds 5mL 2% foetal calf serum (V/V), put 26 ℃ of cultivations, until obvious cytopathic effect appears in cell, the collecting cell lesion material, in-80 ℃ to room temperature (20-25 ℃) multigelation 3 times, the centrifugal 30min of 5000r/min, get supernatant 250 μ l, with lS(Invitrogen) reagent extracts RNA to specifications, finally is dissolved in 50 μ l aqua sterilisas, and-80 ℃ save backup.
Two, the reaction system of RT-LAMP amplification:
Adopt 25 μ l reaction systems, comprising: each 0.8 μ M of inner primer FIP and BIP, each 0.1 μ M of outer primer F3 and B3, dNTPs 1mM, Betaine 0.5M, DTT 4mM, MgCl 2, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l.
Mg wherein 2+concentration be respectively 2,4,6,8 and 10mM.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes is in 64 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, with after 2% agarose gel electrophoresis, be placed in the gel imaging system imaging, electrophoresis Image Display Mg 2+concentration while being 8mM result best.
Embodiment 3:
The optimization of SVCV RT-LAMP detection method temperature of reaction:
One, get sample extraction viral RNA to be checked:
Results (preparation method is with embodiment 2) after 90% pathology appears in the EPC cell infected until SVCV, in-80 ℃ to room temperature multigelation 3 times, the centrifugal 30min of 5000r/min, get supernatant 250 μ l, uses lS reagent extracts RNA to specifications, finally is dissolved in 50 μ l aqua sterilisas, and-80 ℃ save backup.
Two, the reaction system of RT-LAMP amplification:
Adopt 25 μ l reaction systems, comprising: each 0.8 μ M of inner primer FIP and BIP, outer primer F3 and B30.1 μ M, dNTPs1mM, Betaine 0.5M, DTT 4mM, MgCl 28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes was placed in respectively 60,61,62,63,64,65 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, with after 2% agarose gel electrophoresis, be placed in the gel imaging system imaging, during 64 ℃ of electrophoresis Image Displays, result is best.
Embodiment 4:
The specificity of SVCV RT-LAMP detection method
One, get sample extraction viral RNA to be checked:
SVCV(Zhangs friend, Liu's Polygonum, Chen Xiaoxuan, Deng. the preparation of SVCV monoclonal antibody and CHARACTERISTICS IDENTIFICATION thereof [J], China's Preventive Veterinary Medicine newspaper, 2011, 33(4): EPC cell 305-308.) infected, CRV(once made the soldier, Xu advances, Li Yanqiu, Deng. the separation of channel catfish hemorrhagic disease cause of disease reovirus and evaluation [J], the virus journal, 2009, 25(6): CCO cell 460-466.) infected, the CIK cell that GCRV-104 strain (CCTCC NO:V201217) is infected occurs gathering in the crops (the same SVCV of the preparation method of above-mentioned virus) after 90% pathology, in-80 ℃ to room temperature multigelation 3 times, the centrifugal 30min of 5000r/min, get supernatant 250 μ l, with lS reagent extracts RNA to specifications, finally is dissolved in 50 μ l aqua sterilisas, and-80 ℃ save backup.
Two, the reaction system of RT-LAMP amplification:
Adopt 25 μ l reaction systems, comprising: each 0.8 μ M of inner primer FIP and BIP, outer primer F3 and B30.1 μ M, dNTPs1mM, Betaine 0.5M, DTT 4mM, MgCl 28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes is in 64 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, with after 2% agarose gel electrophoresis, be placed in the gel imaging system imaging, electrophoresis picture show needle can guarantee the specific detection to SVCV to the special RT-LAMP primer sets of SVCV design, only detect SVCV, and GCRV-104, CRV and contrast all can't detect (Fig. 1).
SEQUENCE LISTING
<110 > Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120 > a kind of SVCV RT-LAMP detection kit and detection method
<130 > a kind of SVCV RT-LAMP detection kit and detection method
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
<213 > SVCV
<400> 1
GGGATAGCTT CGGACACAAG 20
<210> 2
<211> 20
<212> DNA
<213 > SVCV
<400> 2
CCATCAGCAA AGTCCGGTAT 20
<210> 3
<211> 46
<212> DNA
<213 > SVCV
<400> 3
GTTTCCCATC TGGTAGCCCG TCTTTTAGAT CGGGCCTTTT AACCTG 46
<210> 4
<211> 44
<212> DNA
<213 > SVCV
<400> 4
AGGTGAGTGC TGAGGATGAT GCTTTTTTGC CCTTCCCACT CTGT 44

Claims (5)

1. a SVCV RT-LAMP detection kit, is characterized in that, comprises following composition:
This test kit comprises following composition: 10 * ThermoPol Reaction damping fluid; bstarchaeal dna polymerase; DNTPs; The AMV reversed transcriptive enzyme; Outer primer F3 and B3, inner primer FIP and BIP, Betaine, MgCl 2, DTT, 1000 * SYBR Green I;
F3:5 - GGGATAGCTTCGGACACAAG - 3
B3:5 - CCATCAGCAAAGTCCGGTAT -3
FIP:5 - GTTTCCCATCTGGTAGCCCGTCTTTTAGATCGGGCCTTTTAACCTG - 3
BIP:5 - AGGTGAGTGCTGAGGATGATGCTTTTTTGCCCTTCCCACTCTGT- 3
2. utilize a kind of SVCV RT-LAMP detection kit claimed in claim 1 to detect the method for SVCV, the steps include:
1), obtaining of viral RNA:
Adopt TRIzol or TRIzol lS reagent or post absorb-elute method are extracted the total RNA of sample;
2), RT-LAMP amplification:
Adopt 25 μ l reaction systems: each 0.8 μ M of inner primer FIP and BIP, each 0.1 μ M of outer primer F3 and B3, dNTPs1mM, Betaine0.5M, DTT4mM, MgCl 22-10mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 10 * ThermoPol Reaction Buffer, 2.5 μ l, select 55-65 ℃ and within 30-120 minute, carry out the RT-LAMP reaction after, 80 ℃ of deactivations 2 minutes;
3), detected result judges, adopts one of following three kinds of modes:
When A, generation amplified reaction, the pyrophosphate ion of separating out from dNTPs and the magnesium ion reaction solution form white magnesium pyrophosphate precipitation, by naked eyes, judge: detector tube occurs obviously muddy positive, has no muddiness negative;
The reaction tubes of B, every 25 μ l systems adds 1000 * SYBR Green I 1-2 μ l, 1-5 min observations, and it is green positive that reaction solution turns, and keeps orange negative;
C, get amplified production, after the agarose gel electrophoresis with 2% w/v, be placed in the gel imaging system imaging, electrophoresis Image Display LAMP characteristic scalariform band, result is positive; As without any band, result is negative.
3. a kind of SVCV RT-LAMP detection kit according to claim 2 detects the method for SVCV,, it is characterized in that: MgCl 2concentration be 8mM.
4. a kind of SVCV RT-LAMP detection kit according to claim 2 detects the method for SVCV,, it is characterized in that: temperature of reaction is 64 ℃.
5. a kind of SVCV RT-LAMP detection kit according to claim 2 detects the method for SVCV,, it is characterized in that: template ribonucleic acid carries out pre-treatment in 95 ℃ are placed on ice bath in 5 minutes.
CN2012104030539A 2012-10-19 2012-10-19 RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus) Pending CN102876811A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105002298A (en) * 2015-05-21 2015-10-28 上海市水产研究所 Fluorescent quantitative PCR detection method of spring viraemia of carp virus
CN107267668A (en) * 2017-08-01 2017-10-20 北京科弘生物技术有限公司 Ring mediated isothermal amplification combination lateral flow test strips method detects SVCV
CN109628640A (en) * 2018-12-29 2019-04-16 中国水产科学研究院珠江水产研究所 A kind of RPA-LFD primer, method and the kit of quick detection huichun viremia virus

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105002298A (en) * 2015-05-21 2015-10-28 上海市水产研究所 Fluorescent quantitative PCR detection method of spring viraemia of carp virus
CN105002298B (en) * 2015-05-21 2018-08-31 上海市水产研究所 A kind of fluorescent quantitative PCR detection method of huichun viremia virus
CN107267668A (en) * 2017-08-01 2017-10-20 北京科弘生物技术有限公司 Ring mediated isothermal amplification combination lateral flow test strips method detects SVCV
CN109628640A (en) * 2018-12-29 2019-04-16 中国水产科学研究院珠江水产研究所 A kind of RPA-LFD primer, method and the kit of quick detection huichun viremia virus

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Application publication date: 20130116