CN102703608A - Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses - Google Patents
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses Download PDFInfo
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Abstract
The invention discloses a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and a detection method for grass carp reoviruses (GCRV), and belongs to the technical field of the detection of fish viruses. The RT-LAMP assay kit for the grass carp reoviruses comprises the following ingredients: 1*ThermoPol Reaction Buffer, Bacillus stearothermophilus deoxyribonucleic acid (BstDNA) polymerase, deoxyribonucleotide triphosphate (dNTPs), AMV reverse transcriptase, outer primers (F3 and B3), inner primers (FIP and BIP), Betaine, MgCl2, dithiothreitol (DTT) and 1,000*SYBR Green I. The assay kit has the characteristics of simpleness, convenience, quickness and high specificity and sensitivity; GCRV-104 in a sample can be accurately detected within 2 hours only by using a water bath kettle or metal bath; and the assay kit can detect grass carp tissues infected by the GCRV-104 and cells (such as cytokine-induced killer (CIK) cells) infected by the GCRV-104, and is applicable to the on-site rapid detection of the GCRV-104.
Description
Technical field
The invention belongs to fish virus detection techniques field, be specifically related to a kind of GCRV RT-LAMP detection kit, also relate to a kind of detection method of GCRV.
Background technology
Hemorrhagic disease of grass carp is infectivity, pathogenic all very strong a kind of serious virus disease, main harm grass carp fingerling.Found in the Shekou, Hubei first in 1972, and the called after hemorrhagic disease of grass carp.Confirmed through electron microscopic observation that this cause of disease was a virus first in 1978.Nineteen eighty-three is isolated explosive grass carp hemorrhage disease pathogen first, and identifies that according to researchs such as its morphology, physicochemical properties this virus is reovirus.The 5th meeting of ICTV in 1991 should the virus definite designation be that (Grass carp reovirus GCRV), and includes Reoviridae (Reoviridae) Aquareovirus (Aquareovirus) in to GCRV.GCRV is the first strain fish virus that isolated in China is identified, existing now data confirms that this virus is the strongest strain of virulence in the Aquareovirus, present domestic more than 20 strain isolated of having reported.
Method to the detection of GCRV is a lot; Electron microscopic observation is the most direct method; Cell cultures isolated viral, plaque test, viral nucleic acid polyacrylamide gel electrophoresis classical ways such as (PAGE) be complicacy comparatively, detects GCRV the most frequently used still immunological detection method and RT-PCR method etc. at present both at home and abroad.Immunological method has much room for improvement on susceptibility and specificity, and has problems such as empty window phase, cross reaction, serotype difference.The RT-PCR method operates complicated, and instrument and personnel are required not to be suitable for field quick detection and basic unit's popularization and application than higher.
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 to target gene; Utilize a kind of active archaeal dna polymerase of strand displacement (Bst archaeal dna polymerase) that has; Under constant temperature fast, efficient, high special, amplified target sequence with sensitivity, reverse transcription loop-mediated constant-temperature amplification (RT-LAMP) is in reaction solution, to add a certain amount of reversed transcriptive enzyme, realizes the step amplification of RNA.Introduce improving one's methods of a pair of ring-type primer, further shorten the reaction times.
Summary of the invention
An object of the present invention is to be to provide a kind of GCRV RT-LAMP detection kit; The S8 gene fragment coding region sequence (HM234682.2) of the GCRV-104 strain of announcing according to GenBank; The online software design Auele Specific Primer of Using P rimerExplorer V4; Utilize the specific region of RT-LAMP technology amplified target gene, GCRV GCRV-104 is carried out rapid detection, have the characteristics of easy, quick, high specific and susceptibility from molecular level.
Another object of the present invention is the detection method that has been to provide a kind of GCRV; Present method only needs a water-bath or metal bath can in 2 hours, accurately detect the GCRV in the sample; Can detect the grass carp tissue that GCRV infects; Can detect the cell (for example CIK cell) that GCRV infects, be highly suitable for the field quick detection of GCRV.
To achieve these goals, the present invention adopts following technical scheme:
A kind of GCRV RT-LAMP detection kit comprises following composition:
This test kit comprises following composition: 1 * 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 '-CAGTGTGATCTCGACTTCCG-3 ' is SEQ ID NO.1;
B3:5 '-AGACCAACGCGTCAATCG-3 ' is SEQ ID NO.2;
FIP:5 '-CGGTCGTCTGACGTACACCGTTTTTTGCCGGCATATGGGGTAA-3 ' is SEQ IDNO.3;
BIP:5 '-AGTTGGGTCAATTGGCTACGGTTTTTAGCACCATGGTACTGTTCG-3 ' is SEQIDNO.4.
A kind of detection method of GCRV the steps include:
1, obtaining of viral RNA:
Using
or
LS reagent or adsorption column elution method to extract a sample of total RNA.
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, dNTPs1mM, Betaine0.5M, DTT4mM, MgCl
22-10mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.Select the time range of 55-65 ℃ TR and 30-120 minute to carry out the RT-LAMP reaction, 80 ℃ of deactivations 2 minutes.
3, detected result is judged, carries out result's judgement with one of following three kinds of methods:
When 1) amplified reaction taking place, the pyrophosphate ion and the mg ion the reaction solution of separating out from dNTPs form white magnesium pyrophosphate deposition, and judge through naked eyes: detector tube occurs obviously muddy positive, does not see muddy negative.
2) reaction tubes of per 25 μ l systems adds 1000 * SYBR Green I (Invitrogen) 1-2 μ l, the 1-5min observations, and it is green positive that reaction solution turns, and keeps orange negative.
3) get amplified production, behind the agarose gel electrophoresis with 2% (w/v), place gel imaging system to form images, the electrophoresis picture shows LAMP characteristic scalariform band, and then the result is positive; As do not have any band, then the result is negative.
A kind of detection method of GCRV (top condition) 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, template ribonucleic acid can increase the sensitivity of detection 95 ℃ of pre-treatment that place ice bath after 5 minutes rapidly.
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, dNTPs1mM, Betaine0.5M, DTT4mM, MgCl
28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.Reaction tubes carried out RT-LAMP reaction in 60 minutes in 63 ℃ of incubations, 80 ℃ of deactivations 2 minutes.
3, detected result is judged, carries out result's judgement with one of following three kinds of methods:
When 1) amplified reaction taking place, the pyrophosphate ion and the mg ion the reaction solution of separating out from dNTPs form white magnesium pyrophosphate deposition, and judge through naked eyes: detector tube occurs obviously muddy positive, does not see muddy negative.
2) reaction tubes of per 25 μ l systems adds 1000 * SYBR Green I (Invitrogen) 1-2 μ l, the 1-5min observations, and it is green positive that reaction solution turns, and keeps orange negative.
3) get amplified production, behind the agarose gel electrophoresis of (w/v, below identical), place gel imaging system to form images with 2%, the electrophoresis picture shows LAMP characteristic scalariform band, and then the result is positive; As do not have any band, then the result is negative.
Compared with prior art, the present invention has the following advantages:
1, the invention discloses a kind of GCRV RT-LAMP detection kit, is for the first time the LAMP technology to be applied to detect GCRV;
2, specificity is good, can effectively detect GCRV-104 virus;
3, rapidly and efficiently, about 1 hour of detection time, be highly suitable for the field quick detection of GCRV;
4, do not need special reagent and equipment, it is low to detect cost
5, identify simply, form white magnesium pyrophosphate deposition through the pyrophosphate ion and the mg ion the reaction solution of separating out from dNTPs, but through naked eyes with regard to judged result, through adding 1000 * SYBR Green I, the sensitivity that can improve the result.
Description of drawings
Fig. 1 is the specific synoptic diagram of a kind of GCRV (GCRV-104) RT-LAMP detection method
Swimming lane from left to right is followed successively by total RNA of the blank of water, normal CIK cell total rna, SVCV infection EPC cell, total RNA, the DL2 of CIK cell, 000 Marker are infected in total RNA, GCRV-985 strain and 104 strains of CRV infection CCO cell.
Fig. 2 is the synoptic diagram as a result that a kind of GCRV (GCRV-104) RT-LAMP detects the grass carp tissue
Swimming lane from left to right is followed successively by DL2, and total RNA of CIK cell is infected in total RNA of the blank of 000 Marker, water, 6 grass carp tissues, GCRV-104 strain.
Embodiment
Following instance further specifies the present invention, but should not regard limitation of the present invention.
Embodiment 1:
A kind of GCRV RT-LAMP detection kit comprises following composition:
It comprises following composition this test kit: 1 * 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’-CAGTGTGATCTCGACTTCCG-3’;
B3：5’-AGACCAACGCGTCAATCG-3’;
FIP：5’-CGGTCGTCTGACGTACACCGTTTTTTGCCGGCATATGGGGTAA-3’;
BIP：5’-AGTTGGGTCAATTGGCTACGGTTTTTAGCACCATGGTACTGTTCG-3’。
Embodiment 2:
The Mg of GCRV (GCRV-104) RT-LAMP detection method different concns
2+Optimization
One, get sample extraction viral RNA to be checked:
Doubtful trouble hemorrhagic disease of grass carp grass carp appearance is gathered in the literary composition village in Jianglin County, Jingzhou City, Hubei Province; The viscera tissues such as liver,spleen,kidney of getting the disease fish are in aseptic petridish; With aseptic eye scissors it is shredded; The PBS that adds 10 times of volumes (V/W) grinds to form tissue homogenate together with organizing fragment to change in the lump in the glass homogenizer under the condition of ice bath.With tissue homogenate in-80 ℃ freezing, room temperature (20-25 ℃, identical up and down) is melted down, so multigelation is 3 times.With the centrifugal 30min of 5000 * g, supernatant is with 0.22 μ m filter filtration sterilization under 4 ℃ of conditions.Get the tissue homogenate of 1mL, in the CIK cell that 10 μ g help infectious agent Polybrene to be inoculated into to grow to confluent monolayer, place 28 ℃ of incubators to adsorb 1h, during every 20min reciprocal inclination culturing bottle (5-15) for several times gently, be beneficial to evenly absorption of virus.After absorption finishes, discard viral liquid, every bottle adds people 5mL cell maintenance medium (the MEM substratum that contains 2% serum), places 25 ℃ of incubators to continue to cultivate, day by day observation of cell pathology situation.When 70%-80% CPE appears in cell monolayer, the harvested cell culture, freeze thawing is 2 times under the 80 ℃-room temperature condition of ﹣, under 4 ℃ of conditions, with the centrifugal 20min of 4000 * g, discards deposition, and it is subsequent use that viral liquid is sub-packed in the frozen pipe-80 ℃ of preservations.The applicant delivers this culture to Chinese typical culture collection center (CCTCC) preservation on April 18th, 2012; Preservation address: Chinese Wuhan Wuhan University; Deposit number: CCTCC NO:V201217, classification name: GCRV GCRV-104.
Cultivate grass carp kidney cell (CIK cell) to confluent monolayer, the sucking-off nutrient solution is 0.1 dosage with infection multiplicity; Inoculation 1mL removes the GCRV cell toxicant material GCRV-104 of cell debris through the centrifugal 5min of 4000r/min, adds the Polybrene (final concentration 10 μ g/ml) of 10 μ L, places 28 ℃ of incubators to adsorb 1h; Make virus absorption onto cell, every 20min rocks culturing bottle once gently in the adsorption process, makes that viral liquid and cell monolayer are full and uniform to be contacted; Behind the absorption 1h, inhale and abandon viral liquid, add the nutrient solution of 5mL 2% foetal calf serum (V/V); Put 28 ℃ of cultivations, tangible cytopathic effect occurs, the collecting cell lesion material until cell; In-80 ℃ to room temperature (20-25 ℃) multigelation 3 times; The centrifugal 30min of 5000r/min gets supernatant 250 μ l, extracts RNA to specifications with
LS (Invitrogen) reagent; Be dissolved in 50 μ l aqua sterilisas at last ,-80 ℃ of preservations are subsequent use.
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, dNTPs1mM, Betaine0.5M, DTT4mM, MgCl
2, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.
Mg wherein
2+Concentration be respectively 2,4,6,8 and 10mM.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes is in 63 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, behind 2% agarose gel electrophoresis, place gel imaging system to form images, the electrophoresis picture shows that the result was best when the concentration of Mg2+ was 8mM.
Embodiment 3:
The optimization of GCRV (GCRV) RT-LAMP detection method temperature of reaction:
One, get sample extraction viral RNA to be checked:
Treat that the CIK cell that GCRV-104 infects occurs gathering in the crops (preparation method is with embodiment 2) after 90% pathology; In-80 ℃ to room temperature multigelation 3 times; The centrifugal 30min of 5000r/min; Get supernatant 250 μ l; Extract RNA to specifications with
LS reagent, be dissolved in 50 μ l aqua sterilisas at last ,-80 ℃ of preservations are subsequent use.
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, Betaine0.5M, DTT4mM, MgCl
28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes places 60,61,62,63,64,65 ℃ of incubations after 60 minutes respectively, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, behind 2% agarose gel electrophoresis, place gel imaging system to form images, the result was best when the electrophoresis picture showed 63 ℃.
Embodiment 4:
The specificity of GCRV (GCRV) RT-LAMP detection method
One, get sample extraction viral RNA to be checked:
Treat that the EPC cell of SVCV infection, the CIK cell that CCO cell, GCRV-985 strain and the GCRV-104 strain of CRV infection are infected occur gathering in the crops (the same GCRV-104 of the preparation method of above-mentioned virus) after 90% pathology; Gather in the crops normal CIK cell contrast simultaneously; In-80 ℃ to room temperature multigelation 3 times; The centrifugal 30min of 5000r/min; Get supernatant 250 μ l; Extract RNA to specifications with
LS reagent, be dissolved in 50 μ l aqua sterilisas at last ,-80 ℃ of preservations are subsequent use.
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, Betaine0.5M, DTT4mM, MgCl
28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes is in 63 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions; Behind 2% agarose gel electrophoresis; Place gel imaging system to form images; The electrophoresis picture shows that the special RT-LAMP primer sets to the GCRV-104 design can guarantee the specific detection to GCRV-104, only detect GCRV-104, and GCRV-985, SVCV, CRV and contrast feminine gender all can't detect (Fig. 1).
Embodiment 5:
The RT-LAMP detection method of a kind of GCRV GCRV-104 the steps include:
One, get sample extraction viral RNA to be checked:
The grass carp tissue of getting the doubtful infection GCRV of 100mg add 1ml
reagent with glass homogenizer in grinding at room temperature; Extract RNA to specifications; Be dissolved in 50 μ l aqua sterilisas at last ,-80 ℃ of preservations are subsequent use.
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, Betaine0.5M, DTT4mM, MgCl
28mM, Bst archaeal dna polymerase 8U, AMV reversed transcriptive enzyme 5U, template ribonucleic acid 5 μ l, 1 * ThermoPol Reaction Buffer.
Three, the reaction conditions of RT-LAMP amplification:
Reaction tubes is in 63 ℃ of incubations after 60 minutes, 80 ℃ of deactivations 2 minutes.
Four, detected result is judged:
Get 8 μ l amplified productions, behind 2% agarose gel electrophoresis, place gel imaging system to form images, the electrophoresis picture shows the contrast establishment, and 3 be positive (Fig. 2) are arranged in 6 fish appearance, and the result who detects with the RT-PCR method is consistent.
SEQUENCE?LISTING
< 110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
< 120>a kind of GCRV RT-LAMP detection kit and detection method
< 130>a kind of GCRV RT-LAMP detection kit and detection method
<160> 4
<170> PatentIn?version?3.1
<210> 1
<211> 20
<212> DNA
< 213>GCRV
<400> 1
cagtgtgatc?tcgacttccg 20
<210> 2
<211> 18
<212> DNA
< 213>GCRV
<400> 2
agaccaacgc?gtcaatcg 18
<210> 3
<211> 43
<212> DNA
< 213>GCRV
<400> 3
cggtcgtctg?acgtacaccg?ttttttgccg?gcatatgggg?taa 43
<210> 4
<211> 45
<212> DNA
< 213>GCRV
<400> 4
agttgggtca?attggctacg?gtttttagca?ccatggtact?gttcg 45
Claims (5)
1. a GCRV RT-LAMP detection kit is characterized in that, comprises following composition:
This test kit comprises following composition: 1 * ThermoPol Reaction Buffer;
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
，-?CAGTGTGATCTCGACTTCCG?-?3
，
B3：5
，-?AGACCAACGCGTCAATCG?-3
，
FIP：5
，-?CGGTCGTCTGACGTACACCGTTTTTTGCCGGCATATGGGGTAA?-?3
，
BIP：5
，-?AGTTGGGTCAATTGGCTACGGTTTTTAGCACCATGGTACTGTTCG?-?3
，。
2. the method with the described test kit detection of claim 1 GCRV the steps include:
1), obtaining of viral RNA:
Adopt TRIzol or TRIzol LS reagent or post absorb-elute method to extract 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, 1 * ThermoPol Reaction Buffer, select 55-65 ℃ with carried out the RT-LAMP reaction in 30-120 minute, 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 and the mg ion the reaction solution of separating out from dNTPs form white magnesium pyrophosphate deposition, and judge through naked eyes: detector tube occurs obviously muddy positive, does not see that muddiness is negative;
The reaction tubes of B, per 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, behind the agarose gel electrophoresis with 2%w/v, place gel imaging system to form images, the electrophoresis picture shows LAMP characteristic scalariform band, and then the result is positive; As do not have any band, then the result is negative.
3. a kind of method that detects GCRV according to claim 2 is characterized in that: MgCl
2Concentration be 8mM.
4. a kind of method that detects GCRV according to claim 2 is characterized in that: temperature of reaction is 63 ℃.
5. a kind of method that detects GCRV according to claim 2 is characterized in that: template ribonucleic acid was placed at 95 ℃ and carries out pre-treatment in the ice bath in 5 minutes.
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| 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 |
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| CN104673935A (en) * | 2015-02-12 | 2015-06-03 | 浙江省淡水水产研究所 | Detection method and detection kit of gene II type grass carp reovirus |
| CN105506177A (en) * | 2015-12-17 | 2016-04-20 | 中国水产科学研究院淡水渔业研究中心 | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof |
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| CN106811550A (en) * | 2017-03-11 | 2017-06-09 | 中国水产科学研究院珠江水产研究所 | A kind of type vaccine strain of GCRV II and street strain's diagnostic primerses and kit and diagnosis detecting method containing it |
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| 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 |
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| CN104450961A (en) * | 2014-11-25 | 2015-03-25 | 浙江省淡水水产研究所 | Kit for detecting grass carp reovirus types I, II and III based on RT-LAMP visualization technology and method for detecting grass carp reovirus types I, II and III |
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Application publication date: 20121003 |