CN101838677B - Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method - Google Patents
Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method Download PDFInfo
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Abstract
The invention belongs to the field of virus detection in the biological technology and particularly relates to a preparation method of a nucleic acid sample for diagnosing a shrimp white spot syndrome virus disease and a method for detecting a shrimp white spot syndrome virus by using the nucleic acid sample subjected to LAMP (Loop-mediated Isothermal Amplification). The method for detecting a shrimp white spot syndrome virus comprises the following steps: taking killed fresh shrimp tissue, slightly shearing the killed fresh shrimp tissue and adding the sheared shrimp tissue into buffer solution III; quickly boiling and centrifuging to obtain supernate as a template for amplifying nucleic acid; after carrying out the LAMP, adding SYBR Green I into the loop-mediated isothermal amplification(LAMP) diluted by thousand folds or ten thousand folds; and judging a detection result by observing whether to exist an excited green fluorescent result through naked eyes under the ultraviolet excitation. The invention has the characteristics of high sensitivity, high specificity, simple and convenient operation, short time consumption, fewer required samples, low cost and the like.
Description
Technical field
The invention belongs to the field of virus detection in biotechnology, be specifically related to a kind of preparation method of shrimp white spot syndrome virus disease nucleic acid sample and utilize nucleic acid samples to detect the method for shrimp white spot syndrome virus after the LAMP amplification.
Background technology
The white spot syndrome virus disease is a kind of potent virus sexually transmitted disease, its cause of disease is white spot syndrome virus (WSSV), WSSV is the virus that a kind of infectivity is extremely strong, lethality rate is high, can infect in one week mortality ratio up to 80%-100%, and its host domain is very extensive, become the disease that there is serious threat in sea, freshwater aquiculture prawn or crayfish, once morbidity just is difficult to accomplish timely treatment, may cause shrimps in culture fulminant death to occur, the serious whole pond that even can cause has no harvest, and causes heavy loss to numerous culturists.WSSV has spreaded all over main prawn culturing countries and regions, Asia at present, and has propagated into North America.In all prawn ' s virus of having reported, this virus virulence is the strongest, harm is maximum, Penaeus vannamei, tigar prawn, japonicus, Penaeus merguiensis de man., penaeus penicillatus, Chinese prawn, brown prawn, pink shrimp, blue prawn, white shrimp, brown shrimp, the new prawn of cutter volume, dusky white prawn, tigar prawn etc., most of cultured prawn all can be infected by WSSV, planktonic organism, sea anemone, oppossum shrimp, white shrimp, shrimp, the thick crab in Tianjin, can detect WSSV in the animals such as Japan large eye crab, crab in the shrimp pond, also examine and be WSSV in copepods and insect (Ephydridae) body, therefore as far back as nineteen ninety-five International Office of Epizootics (OIE), Food and Argriculture OrganizationFAO (FAO) and Asian-Pacific area aquaculture development network center (NACA) just classify it as one of important Viral Diseases of Aquatic Animal cause of disease that needs report.Generally believe both at home and abroad and only have comprehensive prevention could control WSSV.With Operand sterilize culturing pool, to grow shrimp throw something and feed immunostimulant, monitoring culture pond every water-quality guideline and take to clear up in time dead shrimp, sick shrimp with cut-out virus disseminating approach.Generally speaking, adopting the measure that reduces infection chance is that most of raisers are in order to avoid healthy shrimp to infect the main method of WSSV.
The research of relevant WSSV is being deep into molecular level aspect etiology, pathology, epidemiology and diagnostics research, but due to due to many reasons, up to the present can't control the epidemic situation of WSSV fully, can take topmost measure in the face of the propagation of WSSV is to find early and eliminate contagium, the resolute route of transmission that cuts off, particularly in breeding environment, in early days sensitive, easy, fast the diagnosis can be for adopting an effective measure in time, reduce the loss, anti-WSSV breaks out the foundation that science is provided on a large scale.
Ring mediated isothermal amplification method (the LAMP that development in recent years is got up, loop-mediated isothermalamplification), it is a kind of brand-new constant temperature nucleic acid amplification method, the method can be with sensitivity efficient amplified target sequence (Notomi et al. very selectively, 2000), final LAMP product is loop-stem structure and the DNA fragmentation mixture that encircles the Cauliflower spline structures that the target sequence of a series of inverted repeats forms more, manifests the staged collection of illustrative plates that different large communities band forms after electrophoresis on gel.It is few that it has a required instrument: only need a thermostat just passable; Sensitive height: template only need 10 the copy or still less; Specific amplification is strong: four primers that application has comprised six sections are increased by strict order; Detection time is short: general amplification can complete with interior at 60min, than common PCR reaction, also will save time.Therefore LAMP has obtained increasingly extensive application in fields such as the sex identification of the diagnosis of the scientific research of nucleic acid, disease and prevention, animal embryo and genetically modified food detections.Usually in order to use the LAMP method to realize detecting, need to prepare in advance the template for amplification, after the LAMP amplification, also need amplified production is carried out to visual observation, to determine the final amplification of LAMP, this result has represented the net result detected.Tomoya Kono in 2004 out, then extracts the nucleic acid of WSSV by WSSV purifying from kuruma shrimp, utilizes the LAMP technology to be increased to this nucleic acid, with electrophoresis observation LAMP result.Finally confirm that LAMP can be used for the amplification of WSSV corresponding sequence, for the WSSV of shrimp diagnosis lays the foundation.Viruses adsorption technology isolated viral for Lakshmi in 2008 etc., cell culture fluid obtains template by the sucrose gradient ultracentrifugation method, with RT-LAMP, detects CHIK virus.Amplification can, by add SYBR Green I in reaction tubes, judge according to colour developing.Within 2008, the use cortisone acetates such as Yang Qiu woods are induced Pc through subcutaneous injection Wistar rat, collect bronchoalveolar lavage fluid (BALF) through centrifugal, add lysis buffer, purification Pc genomic dna after digestion repeatedly, then use ring mediated isothermal amplification (LAMP) technology for detection Pneumocystis carinii (Pc), the positive Pc detector tube of result is green after colour developing, and it is brown that negative control group all is.Yuan Wen equals to use in 2009 QIAamp viral RNA mini kit to extract viral geneome RNA, sets up reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection technique of Mouse hepatitis virus and carries out Preliminary Applications.When detecting amplification, add the colour developing of SYBR Green I fluorescence dye in the LAMP reaction tubes, visible MHV is positive, and pipe becomes green, and negative tube is still that the dyestuff primary colors is orange.These researchs show, the virus (cause of disease) of using purifying to cross prepares template, can with the naked eye directly judge the amplification of LAMP by add the colour developing of SYBR Green I fluorescence dye in the LAMP reaction tubes after increasing by LAMP, simplify the original step of passing through the electrophoresis observation result.
Obviously in the existing whole process detected based on LAMP, the process that its template preparation and LAMP result are observed presents two classes, one class is by purified virus (or specific object to be detected) in advance, can be prepared by a number of procedures template later, the method that now prepares template can be by test kit or simple boiling method, after the LAMP amplification, can or add the colour developing of SYBR Green I fluorescence dye with the direct observations of naked eyes by electrophoresis, Equations of The Second Kind is purified virus not, but directly will comprise the host and virus carry out common extraction at interior tissue, the special amplification of utilizing LAMP amplification, because be has mixed host's nucleic acid and viral nucleic acid in the template preparation, if directly with visual inspection, differentiate result after adding SYBR Green I fluorescence dye by colour developing, the mixing nucleic acid existed while understanding due to common the extraction, with SYBR Green I, be combined, also can produce fluorescence, therefore can not be by directly with visual inspection, judging the result of amplification after SYBR Green I colour developing, to the amplification accurate result, be to judge by electrophoresis at present.
Yet, the method that the LAMP of the extensive practicality of tool detects should be tool high sensitivity, high specific, short, the characteristics such as cost is low easy and simple to handle, consuming time, therefore, need a kind of simple and practical preparation method of shrimp white spot syndrome virus disease nucleic acid sample of research and shrimp white spot syndrome virus detection method on this basis.
Summary of the invention
The object of the invention is to provide a kind of shrimp white spot syndrome virus diagnosis nucleic acid samples preparation method.
For achieving the above object, the technical solution used in the present invention is: a kind of preparation method of shrimp white spot syndrome virus disease nucleic acid sample, comprise the following steps: the tissue of getting the fresh shrimp of execution, be added in damping fluid III, then 95～100 ℃ of heating, more than 3 minutes, are got the template of supernatant as nucleic acid amplification after centrifugal; Wherein, described damping fluid III consists of: 20mM Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), 10mM Repone K (KCl), percent by volume 0.1% triton x-100 (Triton X-100, TritonX-100), 10mM ammonium sulfate (NH
4)
2sO
4, 2mM sal epsom MgSO
4.
In technique scheme, the tissue of described shrimp can be selected any tissue of shrimp, be not strict with, for example: the shrimp foot (comprise the foot of a chela, step all can, and need not shell, can be processed the tissue with shell, this is that the whole bag of tricks of the prior art is all irrealizable, like this without dissecting shrimp, and the shrimp of having cut a trifle foot can also continue survival, therefore, in actual mechanical process, not necessarily need prawn to put to death processing), the shrimp gill, muscle or other tissue; Every 1g shrimp organization need 0.5～50mL damping fluid III in proportion.
In technique scheme, the preferred boiling water bath heating of type of heating, the time of heating is preferably 3～15 minutes, more preferably 5 minutes.
In technique scheme, centrifugation is preferably: centrifugal 5 minutes of whizzer 5000rpm.
The template of the nucleic acid amplification that adopts technique scheme to obtain can be used for LAMP reaction or PCR reaction, particularly, LAMP reaction or 0.5 μ L that the supernatant liquor 1 μ L that prepared by aforesaid method can be used for 25 μ L cumulative volumes as template can be used for the PCR reaction of 25 μ L cumulative volumes as template.
Another object of the present invention is to provide the above-mentioned template of a kind of employing and carries out the method that ring mediated isothermal amplification (LAMP) detects shrimp white spot syndrome virus.
For achieving the above object, the technical solution used in the present invention is: a kind of shrimp white spot syndrome virus detection method comprises the following steps:
(1) take above-mentioned supernatant liquor as amplification template, according to ordinary method, build the ring mediated isothermal amplification system, described amplification system comprises that damping fluid, amplimer are to, dNTP, Bst archaeal dna polymerase, and every 25 μ L amplification systems also comprise the above-mentioned supernatant liquor of 1 μ L;
(2) carry out loop-mediated isothermal amplification, after having reacted, add SYBR Green I in ring mediated isothermal amplification (LAMP) product to dilution after thousand times to ten thousand times, under ultraviolet excitation, by the direct observation of naked eyes, whether there is the green fluorescence result be inspired to judge actual detected result.
In technique scheme, the specificity conservative region design of described amplimer to the shrimp white spot syndrome viral nucleic acid according to increasing, attested amplification purpose zone can be WSSV234 or WSSV131.
Wherein, enzyme be by reaction mixture in 95 ℃ of denaturation 5min, then add amplification reaction system rapidly to cooled on ice.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1, due to the present invention, in nucleic acid-templated preparation process, adopt damping fluid III to process the shrimp tissue, described damping fluid III not only can not have a negative impact to follow-up LAMP or pcr amplification, all right fully cracking shrimp tissue, be conducive to discharge nucleic acid, nucleic acid is stretched as far as possible, and nucleic acid has relative stability in this solution, be blended in albumen mass-energy wherein in preparing the process of sample simultaneously by inactivation, all compositions that add will can not have a negative impact to follow-up LAMP or pcr amplification, nucleic acid (the nucleic acid that comprises shrimp that comes from the non-WSSV do not removed in the template preparation process, the nucleic acid of other cause of disease etc.) can not affect SYBR GREEN I yet the specific amplification result is produced to the colour developing interference, therefore can guarantee that follow-up detection can be smoothly, fast, carry out with high specificity,
2, the present invention prepares the nucleic acid-templated 15min of being about consuming time, even, when with viral nucleic acid with professional, extracting test kit and directly extract the nucleic acid comparison from sick shrimp tissue, still have advantages of that required sample is few, simple to operate, save time, laborsaving, cost is low;
3, the present invention is prepared can be used for detecting the WSSV template, can directly from sick shrimp tissue, obtain, without the complex process of first virus in sick shrimp being carried out to separation and purification;
4, the template that adopts technical solution of the present invention to prepare, can be used for LAMP and PCR reaction, is particularly useful for LAMP, and advantage such as high sensitivity, high specific, the time of while LAMP is short etc., at this, is retained fully;
5, the template that adopts technical solution of the present invention to prepare, combine use through LAMP, the LAMP amplification is after thousand times to ten thousand times of simple dilutions, can eliminate wherein the colour developing background contamination that the DNA from shrimp causes, therefore still can judge diagnostic result by naked eyes by adding SYBR GREEN I, do not need electrophoresis, thereby the LAMP product that has solved (or particular detection object of the purifying) template of only having the use purified virus could be used the restriction of naked eyes direct-detection after SYBR GREEN I colour developing;
6, adopt the method for the invention to detect the shrimp white spot syndrome virus time used in the 90min left and right, detect and compare with other LAMP, whole diagnosis omnidistance (comprising that sample preparation in early stage, LAMP amplification, the LAMP product detect) time used shortens dramatically, and is suitable for mass detection;
7, the detection method of shrimp white spot syndrome virus of the present invention does not need the PCR instrument, also no longer need electrophoresis apparatus, naturally can not use the poisonous nucleic acid dye of this class of EB yet, only need a thermostat (for example water-bath), a small-sized generic centrifuge and a ultraviolet lamp can complete detection, whole operating process is simple, step is little, and cost is low, is conducive to apply.
The accompanying drawing explanation
Fig. 1 is that in embodiment mono-, different methods carries out LAMP electrophoresis as a result after extracting template; Wherein, 1: by the phenol/chloroform method, extract template; 2-6: use respectively damping fluid III, damping fluid I, damping fluid II, ultrapure water and TE, by quick boiling method, extract template;
Fig. 2 is the SYBR Green I colour developing comparison diagram of the rear LAMP product of dilution in embodiment tetra-, and wherein, up 1-5: the LAMP product for preparing template with healthy shrimp dilutes respectively 10,10
2, 10
3, 10
4, 10
5after SYBR Green I colour developing result; Descending 1-5: the LAMP product for preparing template with the sick shrimp of WSSV dilutes respectively 10,10
2, 10
3, 10
4, 10
5after SYBR Green I colour developing result;
Fig. 3 be in embodiment tetra-by SYBR Green I direct colour developing and electrophoresis method detection LAMP product comparison diagram as a result in pipe, wherein, A: with the SYBR Green I result that directly develops the color in centrifuge tube; B: be corresponding EB dyeing electrophoresis result; 1: with healthy shrimp, prepare template LAMP amplified reaction by 1000 dilutions; 2-4: with sick shrimp, prepare template first through 10
3, 10
2, 10
1the template that doubly dilution is re-used as the LAMP amplification is carried out the colour developing result of 1000 times of dilutions after the LAMP amplified reaction;
Fig. 4 is the SYBR Green I colour developing result of injection group LAMP product under ultraviolet lamp in embodiment six, wherein, and 1-6: after injecting 2.5 μ L viral suspension 4h, 5h, 6h, 12h, 18h and 24h, sample the result that detects WSSV; 7: after injection inactivation virus liquid 24h, sampling detects the result of WSSV;
Fig. 5 is the electrophoresis result of injection group LAMP and a step PCR product in embodiment six; Wherein, 1,2,3,5,7,9,11: after injecting 2.5 μ L viral suspension 4h, 5h, 6h, 12h, 18h, 24h and the LAMP detected result after injection inactivation virus liquid 24h; 4,6,8,10: a step PCR detected result of injection 6h, 12h, 18h, 24h sampling; M:Marker.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment mono-: adopt the different templates preparation method prepare amplification by template and carry out the LAMP amplification, the contrast amplification.
Specific operation process is as follows:
(1) extraction of template DNA:
First kind method, classical phenol/chloroform extraction method: get and confirm by the Procambius clarkii of natural infection WSSV, put to death, fully shred, get flesh tissue 0.1g, add TE 100 μ l, 1. add the saturated phenol of 500 μ l, fully mix 10min, the centrifugal 10min of 10000rpm again, the water intaking phase, 2. after repeating 1., add again 500 μ l phenol/chloroforms (1: 1), after fully mixing, the centrifugal 10min of 10000rpm, the water intaking phase, 3. after adding again 500 μ l chloroforms to mix, the centrifugal 10min of 10000rpm, the water intaking phase, 4. the 3M sodium-acetate that adds 1/10 volume, and 95% ethanol of 2 times of cumulative volumes mixes, the centrifugal 10min of 10000rpm, abandon supernatant, to precipitate with 75% ethanol and clean rear drying, be dissolved in again 50 μ l TE standby as the detection DNA profiling.
The Equations of The Second Kind method, by adding different damping fluids to prepare (being called for short fast cooking method) by quick boiling method: get respectively and confirm to be put to death by the Procambius clarkii of natural infection WSSV, after respectively getting same flesh tissue 0.1g and slightly shredding, add respectively the following liquid blending of 100 μ l, in 100 ℃ of water-bath 5min, then the supernatant after the centrifugal 5min of 5000rpm is standby as the template of LAMP reaction; Five kinds of liquid used, all with the ultrapure configuration of sterilizing, are respectively: TE damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA); Damping fluid I (20mmol/L Tris-HCl, 10mmol/L KCl, 0.1%Triton X-100,1mmol/L EDTA); Damping fluid II (20mmol/L Tris-HCl, 10mmol/L K Cl, 0.1%Triton X-100); Damping fluid III (20mmol/L Tris-HCl, 10mmol/L KCl, 0.1%Triton X-100,10mmol/L (NH
4)
2sO
4, 2mmol/L MgSO
4) and the sterilizing ultrapure water.Wherein the formula provided in the present invention is provided for damping fluid I, II, III.
(2) LAMP amplification
LAMP primer used is: front inner primer (FIP-234) is referring to SEQ ID No.1:5 '-CGCATA TTT CCC TCT ATC GCT ATT ATT TTT TAG CAC AGA TTT TTT GAT-3 '.Rear inner primer (BIP-234) is referring to SEQ ID No.2:5 '-GGT CTG AAA TAT ACA TGGGTG CCT TTT TGA AAA TGG GGT TTA CGA CAA-3 '; Outer primer F
3-234 referring to SEQ ID No.3:5 '-GGT AAA GGA ACG TTT TGT TG-3 ', B
3-234 referring to SEQ IDNo.4:5 '-GCA ATG GGA ATG ATA ACT CTT-3 '.Amplification WSSV ORF234 zone.
The reaction of LAMP is 25 μ L systems, 2.5 10 * buffer of μ L, FIP-234, BIP-234 final concentration are 0.8 μ mol/L, F3-234, B3-234 final concentration are 0.2 μ mol/L, the dNTP final concentration is 0.4 μ mol/L, 1.0 the template of the preparation as stated above of μ L and the Bst archaeal dna polymerase of 8U (1 μ L) (New England Biolabs (Beijing), Ltd.).Enzyme be by reaction mixture in 95 ℃ of denaturation 5min, then to cooled on ice, add rapidly.After reaction solution is mixed in 63 ℃ of water-bath 60min.
(3) reaction product of LAMP detects (EB dyeing) through 2% agarose gel electrophoresis, obtains Fig. 1: result shows and is positive with the LAMP of traditional phenol/chloroform method extracting template have and know the band generation; The LAMP reaction that the template of extracting by fast cooking method is carried out, except using ultrapure water as damping fluid, the template of extracting by fast cooking method is without outside obviously band produces after the LAMP amplification, and with other four kinds of damping fluids, the LAMP result by the prepared template of fast cooking method all is positive; Wherein, with damping fluid III, to prepare by quick boiling method the LAMP effect that DNA profiling carries out best, shows as the distinctive trapezoid-shaped strips brightness of LAMP the highest, the most clear.The DNA of preparation also carries out the result far better (Fig. 1) of LAMP reaction as template than the template with the extracting of traditional phenol/chloroform method in this way, but preparing the required time wants much less (time of extracting DNA by classical phenol/chloroform method is roughly suitable with the time that completes whole testing that the present embodiment provides), and the while operation steps is few, simple to operate, with low cost.
Embodiment bis-: with damping fluid III, through fast cooking method, prepare the template special zone in WSSV ORF 234 and WSSV ORF 131 of increasing respectively.
Specific operation process is as follows:
(1) extraction of template DNA: in embodiment mono-, template prepared after boiling soon with damping fluid III by the sick shrimp tissue of the WSSV of Macrobrachium rosenbergii is as mother liquor, after 10 times of serial gradient dilutions, as template, be respectively used to LAMP amplification WSSV ORF 234 and WSSV 131 again.
(2) primer of LAMP amplification WSSV ORF 234 is FIP-234, BIP-234, F described in embodiment mono-
3-234, B
3-234, the primer of amplification WSSV ORF 131 is: FIP-131 is referring to SEQ ID No.5 (5 '-CCA GTG CAC TAT TCC CAT TAG AAG GTT TTA CCA CTC CCT TCATTA TTG TC-3 '), BIP-131 is referring to SEQ ID No.6 (5 '-CCT TTT GCC CCT TCAGCT GA TTT TCT GAG CCA GGT GTT CTG-3 '), F3-131 is referring to SEQ ID No.7 (5 '-TGT GGA TGA TTA TCC TGT GTT-3 '), B3-131 is referring to SEQ ID No.8 (5 '-TCT CTC TGG TGA ACC CAT-3 ').
The LAMP amplification:
Amplification WSSV ORF 234 is with embodiment mono-;
The method of amplification WSSV ORF 131 is with reference to embodiment mono-, and except primer replaces respectively FIP-234, BIP-234, F3-234, B3-234 with FIP-131, BIP-131, F3-131, B3-131, with 63 ℃ of reaction 75min differences, outer all the other are all identical.
(3) observation of LAMP result is with embodiment mono-.
Result shows, want high 10 times by the efficiency ratio for WSSV ORF 234 zone amplifications by the zone for WSSVORF 131, but the two is all very sensitive, and the template that will prepare by fast cooking method is through 10
11doubly after dilution, be used further to the LAMP amplification, all can obtain satisfied result.
Embodiment tri-: by the susceptibility of LAMP, a step PCR and nest-type PRC detection WSSV, compare respectively
(1) preparation of template DNA: by the sick shrimp of 0.1g Procambius clarkii WSSV organize slightly shred after with damping fluid III 100 μ l in 100 ℃ of water-bath 5min, then the supernatant after the centrifugal 5min of 5000rpm is standby as the template of LAMP reaction.
(2) LAMP amplification WSSV ORF 234, and method is with embodiment mono-.
(3) to detect two primer sequences of WSSV:WSSV ORF 234 1 step PCR be respectively P1 to the step PCR of WSSV ORF 234 referring to SEQ ID No.9 (5 '-CCG AAT TCA CCA TGG AGTATA TAG GGG-3 '), P2 referring to SEQ ID No.10 (5 '-CGA AGC TTG ATA CAG TGACCG TCC CTG-3 ').
The step PCR reaction system of 25 μ L comprises 2.5 μ L 10 * PCR buffer, 2.0 μ L MgCl
2(25mmol/L), be respectively P1, the P2 of 0.25 μ L, the template prepared of 0.5 μ L, 0.5 μ L dNTPs (2.5mmol/L), the Taq archaeal dna polymerase of 1U.Reaction conditions is 94 ℃ of denaturation 5min, then carries out 35 circulations: 94 ℃ of sex change 50s, and 51 ℃ of annealing 1min, 72 ℃ are extended 1min, and last 72 ℃ are extended 10min end reaction again.
(4) nest-type PRC of WSSV ORF 234 detects WSSV: two primers are two regional primer PN1 in an above-mentioned step pcr amplified fragment referring to SEQ ID No.11 (5 '-GGG GTT GAA CATTAC GGG-3 ') and PN2 referring to SEQ ID No.12 (5 '-GAT GAC GAG TGG CTTGCT-3 ').
Get the template of step PCR product 1.0 μ L as second step PCR, with PN1 and PN2 primer, carry out pcr amplification, replace P1, PN2 divided by PN1 in reaction solution and replace outside the P2 primer, other component is identical with the first step PCR; Reaction conditions substitutes outside 51 ℃ of annealing 1min in the first step PCR divided by 51 ℃ of annealing 30s, and all the other steps are identical.
(5) observation of detected result: with embodiment mono-.
Result shows can be from diluting 10 by the LAMP method
12wSSV detected in template doubly.During one step pcr amplification, minimum can only be from diluting 10
4wSSV detected in template doubly.Nest-type PRC can be from diluting 10
8wSSV detected in template doubly, and template is diluted 10
9doubly, nest-type PRC also can't amplify the band that theoretical value is the 400bp left and right.
Embodiment tetra-:
The preparation of comparison and detection template: prepare amplification template with healthy Procambius clarkii and the sick shrimp of Procambius clarkii WSSV by the method in embodiment tri-respectively, the template wherein prepared with healthy shrimp is as negative control.
The LAMP amplification: negative control is used undiluted template to be increased, and after the template prepared with sick shrimp is diluted respectively 10,100,1000 times again, for the LAMP amplification, all the other are with embodiment mono-.
The fluorescence observation that directly develops the color: the LAMP amplified production is got respectively to 25 μ L after ten, hundred, thousand, ten thousand and 100,000 times of gradient dilutions, 100 * SYBR Green the I that adds 0.5 μ L, whether have green fluorescence show, obtain Fig. 2 if observing them after the standing 5min of room temperature under ultraviolet lamp; And with the electrophoresis the result.
The electrophoresis observation checking of LAMP result: with embodiment mono-, obtain Fig. 3.
Result shows: when the negative control template, after the LAMP amplification, after dilution more than thousand times, background colour disappears.After the sick shrimp of WSSV is increased with same LAMP, still can send obvious fluorescence (Fig. 2) after 100,000 times of dilutions, explanation is without diluting owing to there being background, can't use after SYBR GREEN I colour developing directly with the unaided eye discrimination result, dilution can be eliminated interference fluorescence, and the dilution in certain limit simultaneously can not have influence on the accurate differentiation to the LAMP positive findings.When using diluted template to carry out the LAMP reaction, reaction solution, through dilution more than thousand times, can accurately show result too afterwards.Electrophoresis result is with direct colour developing result fit like a glove (Fig. 3).
Embodiment five:
The preparation of comparison and detection template: prepare amplification template with healthy Penaeus vannamei, the sick shrimp of Penaeus vannamei WSSV by the method in embodiment tri-respectively.
LAMP amplification: with embodiment mono-.
Add SYBR GREEN I colour developing after the LAMP amplification: with embodiment tetra-.
The electrophoresis observation checking of LAMP result: with embodiment mono-.
Result shows: when the negative control template, after the LAMP amplification, after thousand times of dilutions, background colour disappears.The sick shrimp of WSSV still can send obvious fluorescence after increasing with same LAMP after 100,000 times of dilutions.Explanation after thousand times or ten thousand times of dilutions, can accurately be differentiated the detected result to WSSV by the reacted reaction solution of LAMP with the result of fluorescence developing.
Embodiment six: with damping fluid III by the standby template of fast boiling, LAMP amplification, the direct early detection artificial challenge Procambius clarkii WSSV that develops the color;
The sick shrimp of artificial challenge and contrast shrimp: the sick shrimp of Procambius clarkii of taking from right infection WSSV, execution shreds, and gets shrimp and organizes about 1.0g, after fully grinding (with the rifle head, be not obstructed and be as the criterion) on ice, 3000rpm, centrifugal 5min gained supernatant is the healthy shrimp of injection viral suspension used; The viral suspension of inactivation be by above-mentioned viral suspension 100 ℃ boil 5min after gained, and for control group.Injected dose is that every shrimp is injected 2.5 μ L.
Sampling: 4h, 5h after the injecting virus suspension, 6h, 12h, 18h and 24h be the trifle appendage of clip artificial challenge shrimp respectively, by the method for embodiment tri-, prepares amplification template.The contrast shrimp, with after injecting inactivation viral suspension 24h, samples with the same manner.
LAMP amplification: with embodiment mono-, obtain Fig. 4.
The LAMP amplification detects: whether by 10000 times of dilutions after the LAMP reaction, get 100 * SYBR Green I that diluent 25 μ L add 0.5 μ L, observing them after the standing 5min of room temperature under ultraviolet lamp has green fluorescence to show.LAMP reaction solution with the end dilution carries out electrophoresis (EB dyeing) simultaneously, to be verified by classical way.
One step pcr amplification and result detect: with above-mentioned preparation template, with the method for a step PCR in embodiment tri-, increased, pcr amplification product, by electrophoresis (EB dyeing) observation, obtains Fig. 5.
Result shows: observe and find 4h after infection by direct colour developing with the LAMP amplification, the method for there is no detects WSSV, but, after having infected 5h and reaching, can from the artificial challenge shrimp, WSSV be detected.And contrast shrimp result does not have green fluorescence to occur, show detected result negative (Fig. 4); To after injecting virus suspension 24h, just WSSV can be detected with a step PCR; The electrophoresis result of LAMP amplified production presents (Fig. 5) in full accord with direct fluorescence developing observations.
<110 > University Of Suzhou
<120 > preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detect
Method
<160>12
<210>1
<211>48
<212>DNA
<213 > artificial sequence
<400>1
CGCATATTTC?CCTCTATCGC?TATTATTTTT?TAGCACAGAT
TTTTTGAT?48
<210>2
<211>48
<212>DNA
<213 > artificial sequence
<400>2
GGTCTGAAAT?ATACATGGGT?GCCTTTTTGA?AAATGGGGTT
TACGACAA?48
<210>3
<211>20
<212>DNA
<213 > artificial sequence
<400>3
GGTAAAGGAA?CGTTTTGTTG?20
<210>4
<211>21
<212>DNA
<213 > artificial sequence
<400>4
GCAATGGGAA?TGATAACTCT?T?21
<210>5
<211>50
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<400>5
CCAGTGCACT?ATTCCCATTA?GAAGGTTTTA?CCACTCCCTT
CATTATTGTC?50
<210>6
<211>41
<212>DNA
<213 > artificial sequence
<400>6
CCTTTTGCCC?CTTCAGCTGA?TTTTCTGAGC?CAGGTGTTCT?G?41
<210>7
<211>21
<212>DNA
<213 > artificial sequence
<400>7
TGTGGATGAT?TATCCTGTGT?T?21
<210>8
<211>18
<212>DNA
<213 > artificial sequence
<400>8
TCTCTCTGGT?GAACCCAT?18
<210>9
<211>27
<212>DNA
<213 > artificial sequence
<400>9
CCGAATTCAC?CATGGAGTAT?ATAGGGG?27
<210>10
<211>27
<212>DNA
<213 > artificial sequence
<400>10
CGAAGCTTGA?TACAGTGACC?GTCCCTG?27
<210>11
<211>18
<212>DNA
<213 > artificial sequence
<400>11
GGGGTTGAAC?ATTACGGG?18
<210>12
<211>18
<212>DNA
<213 > artificial sequence
<400>12
GATGACGAGT?GGCTTGCT?18
Claims (2)
1. a shrimp white spot syndrome virus is diagnosed and is used the nucleic acid samples preparation method, it is characterized in that, comprise the following steps: get the tissue of the fresh shrimp of execution, be added in damping fluid III, then 95～100 ℃ of heating, more than 3 minutes, are got the template of supernatant liquor as nucleic acid amplification after centrifugal; Described damping fluid III consists of: 20mM Tri(Hydroxymethyl) Amino Methane Hydrochloride, 10mM Repone K, percent by volume 0.1% triton x-100,10mM ammonium sulfate, 2mM sal epsom; The template of the nucleic acid amplification made further makes shrimp white spot syndrome virus diagnosis nucleic acid samples through the LAMP amplification.
2. preparation method according to claim 1, is characterized in that: every 1g shrimp organization need 0.5～50mL damping fluid III.
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| CN1936549A (en) * | 2006-08-29 | 2007-03-28 | 中国科学院南海海洋研究所 | Reagent box for detecting prawn leucoplakia syndrome virus using isothermal branch augmentation and method therefor |
| CN101153326A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting shigella |
| CN101418352A (en) * | 2008-12-09 | 2009-04-29 | 苏州大学 | Shrimp white spot syndrome virus detection reagent kit and detecting method |
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| CN1936549A (en) * | 2006-08-29 | 2007-03-28 | 中国科学院南海海洋研究所 | Reagent box for detecting prawn leucoplakia syndrome virus using isothermal branch augmentation and method therefor |
| CN101153326A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting shigella |
| CN101418352A (en) * | 2008-12-09 | 2009-04-29 | 苏州大学 | Shrimp white spot syndrome virus detection reagent kit and detecting method |
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