CN104195270B - The visual quick detection kit of white spot syndrome virus (WSSV) and detection method - Google Patents
The visual quick detection kit of white spot syndrome virus (WSSV) and detection method Download PDFInfo
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- CN104195270B CN104195270B CN201410484127.5A CN201410484127A CN104195270B CN 104195270 B CN104195270 B CN 104195270B CN 201410484127 A CN201410484127 A CN 201410484127A CN 104195270 B CN104195270 B CN 104195270B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses the visual quick detection kit of a kind of white spot syndrome virus (WSSV) and detection method, detection kit comprises: reagent A: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution; Reagent B: the Proteinase K aqueous solution; Reagent C: nucleic acid extraction magnetic bead is resuspended in distilled water; Reagent D: Virahol; Reagent E: aqueous ethanolic solution; Reagent F: sterilizing distilled water; Reagent G: white spot syndrome virus (WSSV) positive plasmid; The detector tube of reagent H is housed: the whole operating process of test kit of the present invention is very easy, the detection of prawn WSSV just can be completed within 1.5-2 hour, and without any need for high-end plant and instrument, only need a water-bath or metal bath just can complete, be adapted at promoting the use of in the process of actual production.
Description
Technical field
The present invention relates to the detection technique of cause of disease in aquaculture, be specifically related to visual quick detection kit and the detection method of white spot syndrome virus (WSSV) (WhiteSpotSyndromeVirus, WSSV).
Background technology
White spot syndrome virus (WSSV) is a kind of double-stranded DNA virus with cyst membrane, causes prawn mortality ratio up to 100%, cause serious financial loss to shrimp culture industry in 5-7 days.The host range of white spot virus infection is very wide, not only infects the prawn of various wild and cultivation, and infects other aquatic crustaceans as crab class, crayfish and lobster etc., particularly serious on the impact of prawn population.Within 2009, prawn white spot syndrome is classified as hydrobiont one class epidemic disease by Ministry of Agriculture's " one, two, three class animal epidemic diseases plant register ".Also there is no effective treatment means at present for prawn white spot syndrome, find early and take the necessary measures that to stop virus transmission be generally acknowledge most effective means.Therefore set up sensitive, accurate, fast, easily white spot syndrome virus (WSSV) detection technique be reduce white spot syndrome outburst important channel.
The conventional detection method of current WSSV has: visual observations method, Histological stain method, electron microscopy, euzymelinked immunosorbent assay (ELISA), PCR and quantifying PCR method.Although visual observations method and the Histological stain method used time shorter, generally can only at viral severe infections, just can make a definite diagnosis when producing lesion tissue, not be suitable for early diagnosis; Although electron microscopy, euzymelinked immunosorbent assay (ELISA) and PCR method etc. are highly sensitive, high specificity, complex operation is complicated, needs expensive laboratory apparatus and technical professional, consuming time longer, limits applying aborning.
Ring mediated isothermal amplification (Loop-mediatedisothermalamplification, LAMP) is a kind of new nucleic acid amplification technologies, under the constant temperature of 60-65 DEG C, just can complete nucleic acid amplification reaction through 30-50 minute.Obvious white tetra-sodium precipitation can be formed in LAMP reaction, by the observation of turbidity or add fluorescence dye, judge reaction result by the change of color.This technology has high specific, high efficiency, the feature such as quick, easy, is widely used in the detection of various pathogenic agent.At present, this technology has been applied to the detection of WSSV, but nucleic acid possibly fully cannot extract and cause false negative by the method due to rapid extraction nucleic acid, and the problem such as the false positive adopting the dyestuff such as SYB-Green to produce, hinder LAMP detection technique applying in prawn culturing process.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of visual quick detection kit of white spot syndrome virus (WSSV) is provided.
Second object of the present invention is to provide a kind of method using the visual quick detection kit of white spot syndrome virus (WSSV) to detect white spot syndrome virus (WSSV).
Technical scheme of the present invention is summarized as follows:
The visual quick detection kit of a kind of white spot syndrome virus (WSSV), comprising:
Reagent A, make by following method: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution, in solution, the grams of cetyl trimethylammonium bromide is 1.5-3g, the concentration of NaCl is 1.3-1.5M, the concentration of ethylenediamine tetraacetic acid (EDTA) is 15-25mM, and the concentration of hydroxymethyl aminomethane-hydrochloric acid is 15-25mM; PH=7.5 is regulated to obtain reagent A;
Reagent B: concentration is the Proteinase K aqueous solution of 20-30mg/ml;
Reagent C: 100mg nucleic acid extraction magnetic bead is resuspended in the distilled water of 1ml;
Reagent D: Virahol;
Reagent E: concentration of volume percent is the aqueous ethanolic solution of 65%-75%;
Reagent F: sterilizing distilled water;
Reagent G: white spot syndrome virus (WSSV) positive plasmid;
The detector tube of reagent H is housed: in detector tube, contain MgSO with 10 times in proportion
4isothermal amplification reactions damping fluid 2.5 μ l, the deoxyribonucleotide 3.5 μ l of 40mM, the MgCl of 25mM
26 μ l, the trimethyl-glycine 4 μ l of 5M, the primer P10.4 μ l of use shown in SEQIDNO.1 of 100 μMs, the primer P20.4 μ l of use shown in SEQIDNO.2 of 100 μMs, the primer P30.2 μ l of use shown in SEQIDNO.3 of 100 μMs, the primer P40.2 μ l of use shown in SEQIDNO.4 of 100 μMs, the primer P50.05 μ l of use shown in SEQIDNO.5 of 100 μMs, the primer P60.05 μ l of use shown in SEQIDNO.6 of 100 μMs, BstDNA polysaccharase 8U, fluorexon dyestuff
1 μ l, sterilizing distilled water 3.7 μ l mixes;
Grinding rod, suction pipe, toothpick, 1.5ml sample hose and magnetic frame.
White spot syndrome virus (WSSV) positive plasmid makes by following method: one section of conserved regions sequence SEQIDNO.7 of pcr amplification white spot syndrome virus (WSSV), to tap rubber after agarose gel electrophoresis purifying, to be connected on pMD-18T carrier and to be transformed in TOP10 cell, order-checking qualification positive colony, positive colony correct for order-checking is again cultivated in LB substratum, extracts plasmid with plasmid extraction kit and be white spot syndrome virus (WSSV) positive plasmid.
Use mentioned reagent box to detect the method for white spot syndrome virus (WSSV), comprise the steps:
The preparation of step one, template
(1) get the young shrimp individuality of prawn or the individuality of juvenile prawn or become the gill filament of shrimp to be organized as sample 20-50mg in the 1.5ml sample hose being called sample hose 1, adding 1/3rd places of reagent A to sample hose 1 with suction pipe;
(2) in sample hose 1, drip 1-3 and drip reagent B, ground by sample with grinding rod, 45-60 minute is placed in 50-60 DEG C of water-bath;
(3) with suction pipe Aspirate supernatant in another 1.5ml sample hose being called sample hose 2; Fully blow and beat reagent C with suction pipe, nucleic acid extraction magnetic bead is suspended, in sample hose 2, instill 1 reagent C;
(4) in sample hose 2, instill 10-15 and drip reagent D, after putting upside down mixing gently, be put on magnetic frame; All to focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(5) in sample hose 2, instill 10-20 and drip reagent E, after fully putting upside down mixing, be put on magnetic frame, focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(6) repeating step (5);
(7) opened by the pipe lid of sample hose 2,2-5 minute is placed in 50-60 DEG C of water-bath;
(8) in sample hose 2, instill 1-3 and drip reagent F, flick bottom sample hose with finger, nucleic acid extraction magnetic bead is fully suspended, sample hose 2 is put on magnetic frame, all focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, 3-5 minute is placed in 90-99 DEG C of water-bath, is put on magnetic frame, room temperature cooling 1-3 minute;
Step 2, LAMP amplification and result judge
The sample supernatant in reagent F, reagent G and sample hose 2 is dipped respectively with 3 toothpicks, be equipped with in the detector tube of reagent H to 3, be placed in 60-65 DEG C of water bath heat preservation 30-45 minute, 90-99 DEG C was taken out after water bath heat preservation 1-3 minute, the color of the reaction solution that detects by an unaided eye immediately; Detector tube containing reagent G is that positive control is shown as fluorescent green, and it is orange-yellow that the detector tube containing reagent F is that blank is shown as, and the reaction solution color of sample compares with positive control and blank, represents result.
Prawn is preferably Environment of Litopenaeus vannamei Low, Chinese prawn, tigar prawn or japonicus.
Advantage of the present invention:
The genome conserved sequence district of white spot syndrome virus (WSSV) that we are preferred devises 6 Auele Specific Primers, and 4 Auele Specific Primers of comparing not only increase specificity and the sensitivity of detection, and greatly shorten the reaction times; Adopt magnetic bead to extract the method for nucleic acid, can fully by the nucleic acid extraction in testing sample out, avoid because nucleic acid extraction not exclusively occurs false-negative situation in the process detected afterwards; Use
fluorexon dyestuff can strictly observe the covered principle of LAMP reaction, avoids the similar dyestuff of other colour developing principle such as SYB-Green and must to uncap after amplified reaction terminates situation about adding again, greatly reduce false positive rate; The whole operating process of this test kit is very easy, the detection of prawn WSSV just can be completed within 1.5-2 hour, and without any need for high-end plant and instrument, only need a water-bath or metal bath just can complete, be adapted at promoting the use of in the process of actual production.
Accompanying drawing illustrates:
The color of Fig. 1 test kit detected result judges.In figure, No. 1 pipe is blank reaction result, and No. 2 pipes are positive control reaction result, and No. 3 pipes are embodiment 5 detected result.
Embodiment
Cetyl trimethylammonium bromide, hydroxymethyl aminomethane-hydrochloric acid is purchased from Shanghai Sheng Gong biotechnology limited-liability company;
Proteinase K is purchased from Tian Gen biochemical technology company limited;
Nucleic acid extraction magnetic bead is purchased from Xin Hai east, Xiamen bio tech ltd;
BstDNA polysaccharase, 10 times containing MgSO
4isothermal amplification reactions damping fluid, the deoxyribonucleotide of 40mM, the MgCl of 25mM
2purchased from NewEnglandBiolabs;
The trimethyl-glycine of 5M is purchased from Sigma;
Fluorexon dyestuff
purchased from Rong Yan bio tech ltd;
Plasmid extraction kit is purchased from Tian Gen biochemical technology company limited;
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment 1
The visual quick detection kit of a kind of white spot syndrome virus (WSSV), comprising:
Reagent A, make by following method: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution, in solution, the grams of cetyl trimethylammonium bromide is 1.5g, the concentration of NaCl is 1.3M, the concentration of ethylenediamine tetraacetic acid (EDTA) is 15mM, and the concentration of hydroxymethyl aminomethane-hydrochloric acid is 15mM; PH=7.5 is regulated to obtain reagent A;
Reagent B: concentration is the Proteinase K aqueous solution of 20mg/ml;
Reagent C: 100mg nucleic acid extraction magnetic bead is resuspended in the distilled water of 1ml;
Reagent D: Virahol;
Reagent E: concentration of volume percent is the aqueous ethanolic solution of 65%;
Reagent F: sterilizing distilled water;
Reagent G: white spot syndrome virus (WSSV) positive plasmid (prepared by embodiment 4); Each reagent above can be placed in Reagent Tube and (also can be placed in detector tube);
The detector tube of reagent H is housed: in detector tube, contain MgSO with 10 times in proportion
4isothermal amplification reactions damping fluid 2.5 μ l, the deoxyribonucleotide 3.5 μ l of 40mM, the MgCl of 25mM
26 μ l, the trimethyl-glycine 4 μ l of 5M, the primer P10.4 μ l of use shown in SEQIDNO.1 of 100 μMs, the primer P20.4 μ l of use shown in SEQIDNO.2 of 100 μMs, the primer P30.2 μ l of use shown in SEQIDNO.3 of 100 μMs, the primer P40.2 μ l of use shown in SEQIDNO.4 of 100 μMs, the primer P50.05 μ l of use shown in SEQIDNO.5 of 100 μMs, the primer P60.05 μ l of use shown in SEQIDNO.6 of 100 μMs, BstDNA polysaccharase 8U, fluorexon dyestuff
1 μ l, sterilizing distilled water 3.7 μ l mixes;
Grinding rod; Suction pipe; Toothpick; 1.5ml sample hose; Magnetic frame; Specification sheets.
Embodiment 2
The visual quick detection kit of a kind of white spot syndrome virus (WSSV), comprising:
Reagent A, make by following method: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution, in solution, the grams of cetyl trimethylammonium bromide is 2.5g, the concentration of NaCl is 1.4M, the concentration of ethylenediamine tetraacetic acid (EDTA) is 20mM, and the concentration of hydroxymethyl aminomethane-hydrochloric acid is 20mM; PH=7.5 is regulated to obtain reagent A;
Reagent B: concentration is the Proteinase K aqueous solution of 25mg/ml;
Reagent C, reagent D are with reagent C, the reagent D of embodiment 1;
Reagent E: concentration of volume percent is the aqueous ethanolic solution of 70%;
Reagent F, reagent G, the reagent F of detector tube with embodiment 1, the reagent G of reagent H are housed, the detector tube of reagent H is housed;
Grinding rod; Suction pipe; Toothpick; 1.5ml sample hose; Magnetic frame; Specification sheets.
Embodiment 3
The visual quick detection kit of a kind of white spot syndrome virus (WSSV), comprising:
Reagent A, make by following method: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution, in solution, the grams of cetyl trimethylammonium bromide is 3g, the concentration of NaCl is 1.5M, the concentration of ethylenediamine tetraacetic acid (EDTA) is 25mM, and the concentration of hydroxymethyl aminomethane-hydrochloric acid is 25mM; PH=7.5 is regulated to obtain reagent A;
Reagent B: concentration is the Proteinase K aqueous solution of 30mg/ml;
Reagent C, reagent D are with reagent C, the reagent D of embodiment 1;
Reagent E: concentration of volume percent is the aqueous ethanolic solution of 75%;
Reagent F, reagent G, the reagent F of detector tube with embodiment 1, the reagent G of reagent H are housed, the detector tube of reagent H is housed;
Grinding rod; Suction pipe; Toothpick; 1.5ml sample hose; Magnetic frame; Specification sheets.
Embodiment 4
White spot syndrome virus (WSSV) positive plasmid makes by following method:
One section of conserved regions sequence SEQIDNO.7 of pcr amplification white spot syndrome virus (WSSV), to tap rubber after agarose gel electrophoresis purifying, to be connected on pMD-18T carrier and to be transformed in TOP10 cell, through the LB culture medium flat plate enlarged culturing containing ammonia benzyl, order-checking qualification positive colony, positive colony correct for order-checking is again cultivated in the LB substratum containing ammonia benzyl, extracts plasmid with plasmid extraction kit and be white spot syndrome virus (WSSV) positive plasmid, as the sun mark of test kit.
Embodiment 5
Use the test kit of embodiment 1 to detect the method for white spot syndrome virus (WSSV), comprise the steps:
The preparation of step one, template
(1) the young shrimp individuality getting Chinese prawn, for sample 20mg is in the 1.5ml sample hose being called sample hose 1, adds 1/3rd places (about two full suction pipe) of reagent A to sample hose 1 with suction pipe;
(2) in sample hose 1, drip 1 reagent B, ground by sample fast with grinding rod, 50 DEG C of water-baths place 45 minutes;
(3) take out sample hose, draw supernatant (noting: do not draw residual sample fragment) liquid in another 1.5ml sample hose being called sample hose 2 with suction pipe; Fully blow and beat reagent C with suction pipe, nucleic acid extraction magnetic bead is suspended, in sample hose 2, instill 1 reagent C;
(4) in sample hose 2, instill 10 reagent D with suction pipe, after putting upside down mixing gently, be put on magnetic frame; All to focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(5) in sample hose 2, instill 10 reagent E with suction pipe, after fully putting upside down mixing, be put on magnetic frame, focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(6) repeating step (5);
(7) opened by the pipe lid of sample hose 2, taking-up in 2 minutes is placed in 50 DEG C of water-baths;
(8) in sample hose 2,1 reagent F is instilled, flick bottom sample hose with finger, nucleic acid extraction magnetic bead is fully suspended, sample hose 2 is put on magnetic frame, all focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, 99 DEG C of water-baths place 3 minutes, are put on magnetic frame, and room temperature cools 1 minute; Draw different reagent and will use suction pipe respectively;
Step 2, LAMP amplification and result judge
Pick the sample supernatant in reagent F, reagent G and sample hose 2 respectively with 3 toothpicks, be equipped with in the detector tube of reagent H to 3, be placed in 60 DEG C of water bath heat preservations 30 minutes, 90 DEG C of water bath heat preservations took out after 1 minute, the color of the reaction solution that detects by an unaided eye immediately; Detector tube containing reagent G is that positive control is shown as fluorescent green, and it is orange-yellow that the detector tube containing reagent F is that blank is shown as, and the reaction solution color of sample compares with positive control and blank, and sample result is shown as fluorescent green, positive, sees Fig. 1.1.5 hours consuming time.
Embodiment 6
Use the test kit of embodiment 2 to detect the method for white spot syndrome virus (WSSV), comprise the steps:
The preparation of step one, template
(1) individuality getting the juvenile prawn of tigar prawn be sample 30mg in the 1.5ml sample hose being called sample hose 1, add 1/3rd places (about two full suction pipe) of reagent A to sample hose 1 with suction pipe;
(2) in sample hose 1, drip 2 reagent B, ground by sample fast with grinding rod, 55 DEG C of water-baths place 55 minutes;
(3) take out sample hose, draw supernatant (noting: do not draw residual sample fragment) liquid in another 1.5ml sample hose being called sample hose 2 with suction pipe; Fully blow and beat reagent C with suction pipe, nucleic acid extraction magnetic bead is suspended, in sample hose 2, instill 1 reagent C;
(4) in sample hose 2, instill 12 reagent D with suction pipe, after putting upside down mixing gently, be put on magnetic frame; All to focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(5) in sample hose 2, instill 15 reagent E with suction pipe, after fully putting upside down mixing, be put on magnetic frame, focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(6) repeating step (5);
(7) opened by the pipe lid of sample hose 2, taking-up in 3 minutes is placed in 55 DEG C of water-baths;
(8) in sample hose 2,2 reagent F are instilled, flick bottom sample hose with finger, nucleic acid extraction magnetic bead is fully suspended, sample hose 2 is put on magnetic frame, all focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, 95 DEG C of water-baths place 4 minutes, are put on magnetic frame, and room temperature cools 2 minutes;
Step 2, LAMP amplification and result judge
Pick the sample supernatant in reagent F, reagent G and sample hose 2 respectively with 3 toothpicks, be equipped with in the detector tube of reagent H to 3, be placed in 63 DEG C of water bath heat preservations 40 minutes, 95 DEG C of water bath heat preservations took out after 2 minutes, the color of the reaction solution that detects by an unaided eye immediately; Detector tube containing reagent G is that positive control is shown as fluorescent green, and it is orange-yellow that the detector tube containing reagent F is that blank is shown as, and the reaction solution color of sample compares with positive control and blank, and sample result is shown as orange-yellow, negative.2 hours consuming time.
Embodiment 7
Use the test kit of embodiment 3 to detect the method for white spot syndrome virus (WSSV), comprise the steps:
The preparation of step one, template
(1) getting japonicus becomes the gill filament of shrimp to be organized as sample 50mg in the 1.5ml sample hose being called sample hose 1, adds 1/3rd places (about two full suction pipe) of reagent A to sample hose 1 with suction pipe;
(2) in sample hose 1, drip 3 reagent B, ground by sample fast with grinding rod, 60 DEG C of water-baths place 60 minutes;
(3) take out sample hose, draw supernatant (noting: do not draw residual sample fragment) liquid in another 1.5ml sample hose being called sample hose 2 with suction pipe; Fully blow and beat reagent C with suction pipe, nucleic acid extraction magnetic bead is suspended, in sample hose 2, instill 1 reagent C;
(4) in sample hose 2, instill 15 reagent D with suction pipe, after putting upside down mixing gently, be put on magnetic frame; All to focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(5) in sample hose 2, instill 20 reagent E with suction pipe, after fully putting upside down mixing, be put on magnetic frame, focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, with suction pipe, supernatant liquor sucking-off is discarded;
(6) repeating step (5);
(7) opened by the pipe lid of sample hose 2, taking-up in 5 minutes is placed in 60 DEG C of water-baths;
(8) in sample hose 2,3 reagent F are instilled, flick bottom sample hose with finger, nucleic acid extraction magnetic bead is fully suspended, sample hose 2 is put on magnetic frame, all focus on after at the bottom of pipe until the nucleic acid extraction magnetic bead in pipe, 90 DEG C of water-baths place 5 minutes, are put on magnetic frame, and room temperature cools 3 minutes;
Step 2, LAMP amplification and result judge
Pick the sample supernatant in reagent F, reagent G and sample hose 2 respectively with 3 toothpicks, be equipped with in the detector tube of reagent H to 3, be placed in 65 DEG C of water bath heat preservations 45 minutes, 99 DEG C of water bath heat preservations took out after 3 minutes, the color of the reaction solution that detects by an unaided eye immediately; Detector tube containing reagent G is that positive control is shown as fluorescent green, and it is orange-yellow that the detector tube containing reagent F is that blank is shown as, and the reaction solution color of sample compares with positive control and blank, and sample result is shown as fluorescent green, positive.2 hours consuming time.
Become the gill filament of shrimp to be organized as sample by the method for embodiment 7 to Environment of Litopenaeus vannamei Low to detect, other same the present embodiment, its results sample result is shown as fluorescent green, positive.
Primer synthesis is completed by the raw work in Shanghai.
Claims (2)
1. the visual quick detection kit of white spot syndrome virus (WSSV), is characterized in that comprising:
Reagent A, make by following method: cetyl trimethylammonium bromide, NaCl, ethylenediamine tetraacetic acid (EDTA) and hydroxymethyl aminomethane-hydrochloric acid is added water to 100ml and obtains solution, in solution, the grams of cetyl trimethylammonium bromide is 1.5-3g, the concentration of NaCl is 1.3-1.5M, the concentration of ethylenediamine tetraacetic acid (EDTA) is 15-25mM, and the concentration of hydroxymethyl aminomethane-hydrochloric acid is 15-25mM; PH=7.5 is regulated to obtain reagent A;
Reagent B: concentration is the Proteinase K aqueous solution of 20-30mg/ml;
Reagent C: 100mg nucleic acid extraction magnetic bead is resuspended in the distilled water of 1ml;
Reagent D: Virahol;
Reagent E: concentration of volume percent is the aqueous ethanolic solution of 65%-75%;
Reagent F: sterilizing distilled water;
Reagent G: white spot syndrome virus (WSSV) positive plasmid;
The detector tube of reagent H is housed: in detector tube, contain MgSO with 10 times in proportion
4isothermal amplification reactions damping fluid 2.5 μ l, the deoxyribonucleotide 3.5 μ l of 40mM, the MgCl of 25mM
26 μ l, the trimethyl-glycine 4 μ l of 5M, the primer P10.4 μ l of use shown in SEQIDNO.1 of 100 μMs, the primer P20.4 μ l of use shown in SEQIDNO.2 of 100 μMs, the primer P30.2 μ l of use shown in SEQIDNO.3 of 100 μMs, the primer P40.2 μ l of use shown in SEQIDNO.4 of 100 μMs, the primer P50.05 μ l of use shown in SEQIDNO.5 of 100 μMs, the primer P60.05 μ l of use shown in SEQIDNO.6 of 100 μMs, BstDNA polysaccharase 8U, fluorexon dyestuff
sterilizing distilled water 3.7 μ l mixes;
Grinding rod, suction pipe, toothpick, 1.5ml sample hose and magnetic frame.
2. the visual quick detection kit of a kind of white spot syndrome virus (WSSV) according to claim 1, it is characterized in that described white spot syndrome virus (WSSV) positive plasmid makes by following method: one section of conserved regions sequence SEQIDNO.7 of pcr amplification white spot syndrome virus (WSSV), to tap rubber after agarose gel electrophoresis purifying, to be connected on pMD-18T carrier and to be transformed in TOP10 cell, order-checking qualification positive colony, positive colony correct for order-checking is again cultivated in LB substratum, extract plasmid with plasmid extraction kit and be white spot syndrome virus (WSSV) positive plasmid.
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| CN105695638A (en) * | 2016-04-28 | 2016-06-22 | 中国科学院南海海洋研究所 | Novel rapid detection kit and method for litopenaeus vannamei DNA virus |
| CN109097498A (en) * | 2018-08-31 | 2018-12-28 | 天津市水生动物疫病预防控制中心 | Koi herpesvirus visualizes quick detection kit |
| CN109337990A (en) * | 2018-11-13 | 2019-02-15 | 天津市水生动物疫病预防控制中心 | One seed shrimp liver sausage born of the same parents worm visualizes quick detection kit |
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| CN102912040A (en) * | 2012-10-31 | 2013-02-06 | 广西壮族自治区兽医研究所 | Loop-mediated isothermal amplification primer for detecting white spot syndrome viruses and application of loop-mediated isothermal amplification primer |
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