CN105039593A - Rapid detection kit for viruses of prawn leukoderma syndrome and application of rapid detection kit - Google Patents
Rapid detection kit for viruses of prawn leukoderma syndrome and application of rapid detection kit Download PDFInfo
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Abstract
The invention relates to a rapid detection kit for viruses of prawn leukoderma syndrome and an application of the rapid detection kit. The rapid detection kit comprises the following components: (a) DNA extracting solution A and DNA extracting solution B; (b) LAMP pre-reaction solution; (c) Bst polymerase; (d) LAMP reaction stabilizing solution; (e) developing liquid; (f) positive control DNA; and (g) negative control DNA. The rapid detection kit is strong in specificity and easy and convenient to operate, has higher sensitivity than a PCR method, and does not need an expensive PCR instrument, so that the cost is reduced, and the rapid detection kit is particularly suitable for a primary-level inspection and quarantine institution. A detection result of the detection method is simple and intuitive, and can be directly observed by naked eyes.
Description
Technical field
The present invention relates to marine organisms Pathogen test technology, specifically a kind of quick detection kit of prawn white spot disease syndrome virus and application thereof.
Background technology
White spot syndrome virus (WSSV) extensively to infect the large-scale baculovirus of one of the decapods crustacean seeds such as shrimp crab, adult, parent, since breaking out in cultured prawn from 1993, worldwide wide-scale distribution, cause cultured prawn mortality, cause huge financial loss to culture fishery.Therefore develop new technology and detect WSSV (WSSV fast, accurately, delicately, WhiteSpotSyndromeVirus), strengthen seed and the detection becoming shrimp quarantine and aquaculture water environment, to prophylaxis of viral infections, cut off virus disseminating, ensure that the healthy and sustainable development of China's shrimp culture industry is significant.
Detected white spot syndrome virus (WSSV) in the past and mainly contain three kinds of ways: 1. electron microscopy, this method cost is high, time-consuming, and sensitivity is low, is not suitable for Site Detection; 2. gene probe method, this method cost is high, complicated operation; 3. polymerase chain reaction method (PCR method), this method is quick compared with first two method, sensitive, but needs expensive PCR instrument.
Chinese patent notification number CN1188531C, on February 9 2005 day for announcing, name is called white spot syndrome virus (WSSV) gene diagnosis kit and detection method.This application case discloses a kind of gene diagnosis kit and detection method of white spot syndrome virus (WSSV), be that main body designs according to two pairs of primers of WSSV gene conserved regions sequences Design, adopt polymerase chain reaction (PCR) technology, qualitative detection is carried out to the specific DNA nucleic acid fragment of white spot syndrome virus.Its weak point is, detects the PCR instrument cost used higher.
Summary of the invention
The object of the invention is to that method in order to solve existing detection white spot syndrome virus is time-consuming, cost is high, the defect of complicated operation and a kind of quick detection kit of prawn white spot disease syndrome virus is provided.
Another object of the present invention is the application of the quick detection kit providing a kind of prawn white spot disease syndrome virus.
To achieve these goals, the present invention is by the following technical solutions:
A quick detection kit for prawn white spot disease syndrome virus, described quick detection kit comprises:
A) DNA extraction liquid A is a a with DNA extraction liquid B;
B) LAMP pre-reaction liquid is a;
C) Bst polysaccharase is a;
D) LAMP stable reaction liquid is a;
E) nitrite ion is a;
F) positive control dna is a;
G) negative control DNA is a;
Wherein, described components b) comprise 2-4mmol/L magnesium sulfate, 10-12mmol/L Repone K, 10-12mmol/L ammonium sulfate, massfraction 1-1.5%TritonX-100,2.2-2.7mmol/LdNTP, 0.2-0.8 μm ol/L primer WSSV-B3 and 20-25mmol/L, the pH of primer WSSV-F3,0.2-0.5 μm ol/L of primer WSSV-BIP, 0.2-0.5 μm ol/L of primer WSSV-FIP, 0.2-0.8 μm ol/L be the Tris-HCl of 8-9;
The nucleotide sequence of above-mentioned primer is as follows: WSSV-FIP:5 '-GCTCTTGTTGTTGTTGTTGTCGCTTTTAAATAGGATGTTGTCTCAACC (SEQIDNO.1); WSSV-BIP:
5’-GCTTTCGCGGATTACCCTGTGGTTTTGAAAGATGTCCTACGACCT(SEQIDNO.2)；WSSV-F3：5’-GACAACACTCTTCTTTCTTGAA(SEQIDNO.3)；
WSSV-B3：5’-CCAAGCTTTTACTTCTTCTTGATTTC(SEQIDNO.4)。
In the technical program, the present invention is according to the gene order of white spot syndrome virus (WSSV) Viral structural protein VP2 5, devise one group and specially can identify six of target DNA not homotactic two inner primers and two outer primers, inner primer comprises positive-sense strand and the antisense strand of target DNA; First one of them inner primer hybridizes with target DNA, strand displacement DNA subsequently synthesizes, in the presence of archaeal dna polymerase with height strand-displacement activity, started by an outer primer, discharge single stranded DNA, and as the template that the DNA started by the inner primer of the other end and outer primer that hybridize to target synthesizes, produce an original stem circular DNA; Inner primer for template, starts the synthesis of strand displacement DNA with original stem circular DNA, produces an original stem circular DNA and a new stem circular DNA having twice stem length; Under constant temperature, inner primer is with stem circular DNA for template, and form multiple stem circular DNA containing target DNA tumor-necrosis factor glycoproteins repeatedly by strand displacement, in one hour, this circulating reaction can make DNA be accumulated to 10
9copy, finally by adding fluorescence dye to observe amplification.
As preferably, component a) in DNA extraction liquid A be Tissue lysates, DNA extraction liquid B is DNA adsorption liquid.
As preferably, amount of component b) Bst polysaccharase is the Bst polysaccharase of every microlitre containing 10-12 activity unit.
As preferably, component d) LAMP stable reaction liquid is mineral oil.
As preferably, component e) fluorescence dye of nitrite ion to be massfraction be 5%SYBRGreenI.
An application for the quick detection kit of prawn white spot disease syndrome virus, comprises the following steps:
A) sample preparation: get 10-50mg prawn tissue, be placed in centrifuge tube, adds the DNA extraction liquid A of 100-120 μ L, smashs 1-2min to pieces, at 98-100 DEG C, boil 8-12min; Then be cooled to room temperature, the centrifugal 3-4min of 10000-12000r/min, get supernatant liquor and be placed in another centrifuge tube, add the DNA extraction liquid B of 45-55 μ L, mixing, obtains template DNA to be measured;
B) nucleic acid amplification: according to the number of detected sample, arranging required LAMP reaction tubes number is n, n is sample number+pipe positive control dna+pipe negative control DNA, the nuclease free aqua sterilisa of the LAMP pre-reaction liquid of 8-9 μ L, the Bst polysaccharase of 1-2 μ L and 12-15 μ L is added in each pipe, mix, the centrifugal 10-15s of 2000r/min, then add the LAMP stable reaction liquid of 25-30 μ L, mix, the centrifugal 10-15s of 2000r/min; In a said n reaction tubes, add template DNA to be measured, positive control dna and negative control DNA respectively and mark, then by reaction tubes isothermal reaction 1-1.5h at 65 DEG C;
C) result detects: after reaction terminates, and takes out reaction tubes, is cooled to room temperature, respectively adds 1-2 μ L nitrite ion in reaction tubes, and mixing is observed.
The invention has the beneficial effects as follows:
1) high specificity of the present invention, simple to operation;
2) there is higher sensitivity compared with PCR method, but do not need expensive PCR instrument, reduce cost, be particularly useful for inspection and quarantine mechanism of basic unit;
3) result detection method simple, intuitive of the present invention, directly can observe with the naked eye detected result.
Embodiment
Below by way of specific embodiment, the present invention will be further explained:
Embodiment 1
An application for the quick detection kit of prawn white spot disease syndrome virus, comprises the following steps:
A) sample preparation: get 10mg prawn tissue, be placed in centrifuge tube, adds the DNA extraction liquid A of 100 μ L, smashs 1min to pieces, at 98 DEG C, boil 8min; Then be cooled to room temperature, the centrifugal 3min of 10000r/min, get supernatant liquor and be placed in another centrifuge tube, add the DNA extraction liquid B of 45 μ L, mixing, obtains template DNA to be measured;
B) nucleic acid amplification: according to the number of detected sample, arranging required LAMP reaction tubes number is n, n is sample number+pipe positive control dna+pipe negative control DNA, the nuclease free aqua sterilisa of the LAMP pre-reaction liquid of 8 μ L, the Bst polysaccharase of 1 μ L and 12 μ L is added in each pipe, mix, the centrifugal 10s of 2000r/min, then add the LAMP stable reaction liquid of 25 μ L, mix, the centrifugal 10s of 2000r/min; In a said n reaction tubes, add template DNA to be measured, positive control dna and negative control DNA respectively and mark, then by reaction tubes isothermal reaction 1-h at 65 DEG C; Wherein, LAMP pre-reaction liquid comprise 20mmol/L, pH be 8 Tris-HCl, 2mmol/L magnesium sulfate, 10mmol/L Repone K, 10mmol/L ammonium sulfate, 1%TritonX-100,2.2mmol/LdNTP, the primer WSSV-FIP of 0.2 μm of ol/L, the primer WSSV-BIP of 0.2 μm of ol/L, 0.2 μm of ol/L the primer WSSV-B3 of primer WSSV-F3 and 0.2 μm ol/L; Bst polysaccharase is the Bst polysaccharase of every microlitre containing 10 activity units; LAMP stable reaction liquid is mineral oil;
The nucleotide sequence of above-mentioned primer is as follows: WSSV-FIP:5 '-GCTCTTGTTGTTGTTGTTGTCGCTTTTAAATAGGATGTTGTCTCAACC (SEQIDNO.1); WSSV-BIP:
5’-GCTTTCGCGGATTACCCTGTGGTTTTGAAAGATGTCCTACGACCT(SEQIDNO.2)；WSSV-F3：5’-GACAACACTCTTCTTTCTTGAA(SEQIDNO.3)；
WSSV-B3：5’-CCAAGCTTTTACTTCTTCTTGATTTC(SEQIDNO.4)。
C) result detects: after reaction result, and take out reaction tubes, be cooled to room temperature, in reaction tubes, respectively add 1 μ L massfraction is the fluorescence dye of 5%SYBRGreenI, and mixing is observed.
Be orange at negative control reaction tubes liquid, under the greeny condition of positive control reaction tubes liquid:
A) measuring samples reaction tubes liquid is in green, and this sample result is that white spot syndrome virus (WSSV) is positive;
B) measuring samples reaction tubes liquid is orange, then can report that white spot syndrome virus (WSSV) assay is for negative;
Embodiment 2
An application for the quick detection kit of prawn white spot disease syndrome virus, comprises the following steps:
A) sample preparation: get 30mg prawn tissue, be placed in centrifuge tube, adds the DNA extraction liquid A of 110 μ L, smashs 2min to pieces, at 99 DEG C, boil 10min; Then be cooled to room temperature, the centrifugal 4min of 11000r/min, get supernatant liquor and be placed in another centrifuge tube, add the DNA extraction liquid B of 50 μ L, mixing, obtains template DNA to be measured;
B) nucleic acid amplification: according to the number of detected sample, arranging required LAMP reaction tubes number is n, n is sample number+pipe positive control dna+pipe negative control DNA, the nuclease free aqua sterilisa of the LAMP pre-reaction liquid of 9 μ L, the Bst polysaccharase of 2 μ L and 14 μ L is added in each pipe, mix, the centrifugal 13s of 2000r/min, then add the LAMP stable reaction liquid of 28 μ L, mix, the centrifugal 12s of 2000r/min; In a said n reaction tubes, add template DNA to be measured, positive control dna and negative control DNA respectively and mark, then by reaction tubes isothermal reaction 1.25h at 65 DEG C; Wherein, LAMP pre-reaction liquid comprise 22mmol/L, pH be 8.2 Tris-HCl, 3mmol/L magnesium sulfate, 11mmol/L Repone K, 11mmol/L ammonium sulfate, 1.25%TritonX-100,2.5mmol/LdNTP, the primer WSSV-FIP of 0.5 μm of ol/L, the primer WSSV-BIP of 0.5 μm of ol/L, 0.3 μm of ol/L the primer WSSV-B3 of primer WSSV-F3 and 0.3 μm ol/L; Bst polysaccharase is the Bst polysaccharase of every microlitre containing 11 activity units; LAMP stable reaction liquid is mineral oil;
The nucleotide sequence of above-mentioned primer is as follows: WSSV-FIP:5 '-GCTCTTGTTGTTGTTGTTGTCGCTTTTAAATAGGATGTTGTCTCAACC (SEQIDNO.1); WSSV-BIP:
5’-GCTTTCGCGGATTACCCTGTGGTTTTGAAAGATGTCCTACGACCT(SEQIDNO.2)；WSSV-F3：5’-GACAACACTCTTCTTTCTTGAA(SEQIDNO.3)；
WSSV-B3：5’-CCAAGCTTTTACTTCTTCTTGATTTC(SEQIDNO.4)。
C) result detects: after reaction result, and take out reaction tubes, be cooled to room temperature, in reaction tubes, respectively add 2 μ L massfractions is the fluorescence dye of 5%SYBRGreenI, and mixing is observed.
Be orange at negative control reaction tubes liquid, under the greeny condition of positive control reaction tubes liquid:
A) measuring samples reaction tubes liquid is in green, and this sample result is that white spot syndrome virus (WSSV) is positive;
B) measuring samples reaction tubes liquid is orange, then can report that white spot syndrome virus (WSSV) assay is for negative;
Embodiment 3
An application for the quick detection kit of prawn white spot disease syndrome virus, comprises the following steps:
A) sample preparation: get 50mg prawn tissue, be placed in centrifuge tube, adds the DNA extraction liquid A of 120 μ L, smashs 2min to pieces, at 100 DEG C, boil 12min; Then be cooled to room temperature, the centrifugal 4min of 12000r/min, get supernatant liquor and be placed in another centrifuge tube, add the DNA extraction liquid B of 55 μ L, mixing, obtains template DNA to be measured;
B) nucleic acid amplification: according to the number of detected sample, arranging required LAMP reaction tubes number is n, n is sample number+pipe positive control dna+pipe negative control DNA, the nuclease free aqua sterilisa of the LAMP pre-reaction liquid of 9 μ L, the Bst polysaccharase of 2 μ L and 15 μ L is added in each pipe, mix, the centrifugal 15s of 2000r/min, then add the LAMP stable reaction liquid of 30 μ L, mix, the centrifugal 15s of 2000r/min; In a said n reaction tubes, add template DNA to be measured, positive control dna and negative control DNA respectively and mark, then by reaction tubes isothermal reaction 1.5h at 65 DEG C; Wherein, LAMP pre-reaction liquid comprise 25mmol/L, pH be 9 Tris-HCl, 4mmol/L magnesium sulfate, 12mmol/L Repone K, 12mmol/L ammonium sulfate, 1.5%TritonX-100,2.7mmol/LdNTP, the primer WSSV-FIP of 0.8 μm of ol/L, the primer WSSV-BIP of 0.8 μm of ol/L, 0.5 μm of ol/L the primer WSSV-B3 of primer WSSV-F3 and 0.5 μm ol/L; Bst polysaccharase is the Bst polysaccharase of every microlitre containing 12 activity units; LAMP stable reaction liquid is mineral oil;
The nucleotide sequence of above-mentioned primer is as follows: WSSV-FIP:5 '-GCTCTTGTTGTTGTTGTTGTCGCTTTTAAATAGGATGTTGTCTCAACC (SEQIDNO.1); WSSV-BIP:
5’-GCTTTCGCGGATTACCCTGTGGTTTTGAAAGATGTCCTACGACCT(SEQIDNO.2)；WSSV-F3：5’-GACAACACTCTTCTTTCTTGAA(SEQIDNO.3)；
WSSV-B3：5’-CCAAGCTTTTACTTCTTCTTGATTTC(SEQIDNO.4)。
C) result detects: after reaction result, and take out reaction tubes, be cooled to room temperature, in reaction tubes, respectively add 2 μ L massfractions is the fluorescence dye of 5%SYBRGreenI, and mixing is observed.
Be orange at negative control reaction tubes liquid, under the greeny condition of positive control reaction tubes liquid:
A) measuring samples reaction tubes liquid is in green, and this sample result is that white spot syndrome virus (WSSV) is positive;
B) measuring samples reaction tubes liquid is orange, then can report that white spot syndrome virus (WSSV) assay is for negative.
SEQUENCELISTING
<110> Zhejiang Marine Development Research Institute
The quick detection kit of a <120> prawn white spot disease syndrome virus and application
<130>ZH10012
<160>4
<170>PatentInversion3.3
<210>1
<211>48
<212>DNA
<213> synthetic
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gctcttgttgttgttgttgtcgcttttaaataggatgttgtctcaacc48
<210>2
<211>45
<212>DNA
<213> synthetic
<400>2
gctttcgcggattaccctgtggttttgaaagatgtcctacgacct45
<210>3
<211>22
<212>DNA
<213> synthetic
<400>3
gacaacactcttctttcttgaa22
<210>4
<211>26
<212>DNA
<213> synthetic
<400>4
ccaagcttttacttcttcttgatttc26
Claims (6)
1. a quick detection kit for prawn white spot disease syndrome virus, is characterized in that, described quick detection kit comprises:
A) DNA extraction liquid A is a a with DNA extraction liquid B;
B) LAMP pre-reaction liquid is a;
C) Bst polysaccharase is a;
D) LAMP stable reaction liquid is a;
E) nitrite ion is a;
F) positive control dna is a;
G) negative control DNA is a;
Wherein, described components b) comprise 2-4mmol/L magnesium sulfate, 10-12mmol/L Repone K, 10-12mmol/L ammonium sulfate, massfraction 1-1.5%TritonX-100,2.2-2.7mmol/LdNTP, 0.2-0.8 μm ol/L primer WSSV-B3 and 20-25mmol/L, the pH of primer WSSV-F3,0.2-0.5 μm ol/L of primer WSSV-BIP, 0.2-0.5 μm ol/L of primer WSSV-FIP, 0.2-0.8 μm ol/L be the Tris-HCl of 8-9;
The nucleotide sequence of above-mentioned primer is as follows: WSSV-FIP:5 '-GCTCTTGTTGTTGTTGTTGTCGCTTTTAAATAGGATGTTGTCTCAACC (SEQIDNO.1); WSSV-BIP:
5’-GCTTTCGCGGATTACCCTGTGGTTTTGAAAGATGTCCTACGACCT(SEQIDNO.2)；
WSSV-F3：5’-GACAACACTCTTCTTTCTTGAA(SEQIDNO.3)；
WSSV-B3：5’-CCAAGCTTTTACTTCTTCTTGATTTC(SEQIDNO.4)。
2. the quick detection kit of a kind of prawn white spot disease syndrome virus according to claim 1, is characterized in that, component a) middle DNA extraction liquid A is Tissue lysates, and DNA extraction liquid B is DNA adsorption liquid.
3., according to the quick detection kit of a kind of prawn white spot disease syndrome virus of claim 1 or 2, it is characterized in that, amount of component b) Bst polysaccharase is every microlitre containing the Bst polysaccharase of 10-12 activity unit.
4. the quick detection kit of a kind of prawn white spot disease syndrome virus according to claim 3, is characterized in that, component d) LAMP stable reaction liquid is mineral oil.
5. the quick detection kit of a kind of prawn white spot disease syndrome virus according to claim 4, is characterized in that, component e) fluorescence dye of nitrite ion to be massfraction be 5%SYBRGreenI.
6. an application for the quick detection kit of prawn white spot disease syndrome virus as claimed in claim 1, is characterized in that, comprise the following steps:
A) sample preparation: get 10-50mg prawn tissue, be placed in centrifuge tube, adds the DNA extraction liquid A of 100-120 μ L, smashs 1-2min to pieces, at 98-100 DEG C, boil 8-12min; Then be cooled to room temperature, the centrifugal 3-4min of 10000-12000r/min, get supernatant liquor and be placed in another centrifuge tube, add the DNA extraction liquid B of 45-55 μ L, mixing, obtains template DNA to be measured;
B) nucleic acid amplification: according to the number of detected sample, arranging required LAMP reaction tubes number is n, n is sample number+pipe positive control dna+pipe negative control DNA, the nuclease free aqua sterilisa of the LAMP pre-reaction liquid of 8-9 μ L, the Bst polysaccharase of 1-2 μ L and 12-15 μ L is added in each pipe, mix, the centrifugal 10-15s of 2000r/min, then add the LAMP stable reaction liquid of 25-30 μ L, mix, the centrifugal 10-15s of 2000r/min; In a said n reaction tubes, add template DNA to be measured, positive control dna and negative control DNA respectively and mark, then by reaction tubes isothermal reaction 1-1.5h at 65 DEG C;
C) result detects: after reaction terminates, and takes out reaction tubes, is cooled to room temperature, respectively adds 1-2 μ L nitrite ion in reaction tubes, and mixing is observed.
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| CN107365858A (en) * | 2017-08-22 | 2017-11-21 | 中国科学院海洋研究所 | Health status LAMP detection primer and system and method are detected after exopalaemon carinicauda infection WSSV |
| CN112853004A (en) * | 2021-03-07 | 2021-05-28 | 珠海市迪奇孚瑞生物科技有限公司 | LAMP detection degenerate primer group, kit and method for white spot syndrome virus |
| TWI784467B (en) * | 2018-10-05 | 2022-11-21 | 福又達生物科技股份有限公司 | Pairs of oligonucleotides and probes for detecting white spot syndrome virus (wssv) in shrimps |
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| CN107365858A (en) * | 2017-08-22 | 2017-11-21 | 中国科学院海洋研究所 | Health status LAMP detection primer and system and method are detected after exopalaemon carinicauda infection WSSV |
| TWI784467B (en) * | 2018-10-05 | 2022-11-21 | 福又達生物科技股份有限公司 | Pairs of oligonucleotides and probes for detecting white spot syndrome virus (wssv) in shrimps |
| CN112853004A (en) * | 2021-03-07 | 2021-05-28 | 珠海市迪奇孚瑞生物科技有限公司 | LAMP detection degenerate primer group, kit and method for white spot syndrome virus |
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