CN101643792A - On-site rapid high-sensitivity detection kit and detection method of taura syndrome virus (TSV) - Google Patents
On-site rapid high-sensitivity detection kit and detection method of taura syndrome virus (TSV) Download PDFInfo
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- CN101643792A CN101643792A CN200910017896A CN200910017896A CN101643792A CN 101643792 A CN101643792 A CN 101643792A CN 200910017896 A CN200910017896 A CN 200910017896A CN 200910017896 A CN200910017896 A CN 200910017896A CN 101643792 A CN101643792 A CN 101643792A
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Abstract
The invention relates to an on-site rapid high-sensitivity detection kit and a detection method of taura syndrome virus (TSV). The on-site rapid high-sensitivity detection kit comprises a sampling tube, a rinsing tube filled with distilled water, a nucleic acid denaturation tube filled with TE buffer liquid, an amplification detection tube filled with amplification reaction liquid and nucleic acid dye, a negative control tube, a positive control tube, a FTA membrane, quick drying liquid, and the like. The detection method has higher specificity, sensitivity and convenience than the TR-PCR detection method and has the advantages of low cost, no toxicity, high safety, convenient use, more accurate and quicker detection and high sensitivity and can replace the prior related detection methods, such as a pathological section method, an electron-microscopic observation method, an antibody detection method and a TR-PCR detection method. The on-site rapid high-sensitivity detection kit and the detection method can be both used in an indoor laboratory and a production site in the field, have significance for strengthening the TSV epidemiological monitoring and the disease control and prevention and have high popularization and application value.
Description
Technical field
The present invention relates to marine organisms cause of disease detection technique, specifically is the prawn Taura syndrome field fast high-sensitive detecting kit and the detection method thereof of (Taura syndrome virus is called for short TSV).
Background technology
The prawn Taura syndrome is a single strand RNA virus, belong to the bicistronic mRNA Viraceae, this virus mainly infects the blue prawn (Penaeus stylirostrus) in Environment of Litopenaeus vannamei Low (PenaeusVannamei) and South America, but also infection of Chinese prawn (Penaeus chinensis), japonicus (Penaeusjaponicus), tigar prawn (Penaeus monodon) etc.; Wherein responsive with Environment of Litopenaeus vannamei Low, mortality ratio is up to 40～95% behind the prawn infective virus, and this virus once caused the massive losses of America shrimp culture industry.Culture the rapid expansion of scale in recent years along with China's Environment of Litopenaeus vannamei Low, the extensive risk that takes place of TSV also increases gradually.We find in carrying out national prawn virus disease epidemiology survey in the laboratory that the ratio that Chinese prawn that culture in the Environment of Litopenaeus vannamei Low of the southern breed of China at present, the north and japonicus infect TSV is all very high.In order to keep away the reproduction of angle China shrimp culture industry eighties of last century white spot syndrome virus nineties outburst tragedy, the prawn peach be should set up as early as possible and sensitivity, field diagnostic technology accurately and rapidly drawn, strengthen prawn seed and the monitoring that becomes shrimp quarantine and aquaculture water environment, with prophylaxis of viral infections, cut off virus disseminating, prevent that the large-scale outbreak of TSV is popular.
At present, TSV detects and also mainly depends on pathological section method, electron microscopic observation method and RT-PCR detection method.The pathological section method can not directly detect virus, can only utilize the histopathology sign of morbidity to detect, and be confined to carry out in the laboratory; Though Electron Microscopy can observe directly the existence of virus particle, its complicated operation, expend time in long, accuracy is low; Be better than the pathological section method though detect TSV with antibody testing method on speed, its sensitivity is lower, does not cause infect or infect extremely early stage as yet at TSV and is difficult to detect with antibody act; The RT-PCR detection method of TSV, though overcome the shortcoming of front several method, under laboratory condition, can realize quick relatively, accurately detection to TSV, but, conventional RT-PCR needs expensive PCR instrument, gel electrophoresis and imaging system because detecting, make the RT-PCR detection method to be difficult to use in on-the-spot rapid detection, this has limited RT-PCR detection method applying aborning greatly.
Carrying out prawn Taura syndrome early detection and taking preventive measures is the effective means that the popular risk of reduction prawn Taura syndrome in the prawn culturing, the morbidity of minimizing prawn cause huge financial loss, so set up simple, quick, high-sensitive inspection method, and the prawn Taura syndrome detection kit that exploitation is suitable for rig-site utilization just seems particularly important.
Summary of the invention
The objective of the invention is will according to for a kind of be suitable for the production scene use, fast, highly sensitive and easy-operating prawn Taura syndrome detection method, and with the detection method stdn, simultaneously detection reagent is concentrated in the test kit of standard, to overcome the deficiencies in the prior art, make the detection of TSV more accurate, sensitive, quick, safety and convenient.
At first, utilization of the present invention is at 4 special primers of conservative section design in the TSV capsid protein gene, a kind of ThermoScript II and a kind ofly have an active archaeal dna polymerase of strand displacement, need not separately purpose nucleic acid (RNA) to be carried out reverse transcription, insulation for some time can be finished reverse transcription and the amplification to purpose nucleic acid synchronously under steady temperature, realizes the quick and highly sensitive detection to TSV.In order further to shorten detection time, eliminate contingent pollution in the testing process, to simplify and detect step, test experience can be carried out in the production scene, the present invention optimizes the laboratory detection method of TSV.Be equipped with the FTA blocking in the standard step of sample nucleic acid, needing and just can be carried out rinsing then by the FTA diaphragm of sample homogenization liquid wetting dry at least one hour at room temperature; The present invention is by dripping wetting ability and volatile alcohols material (mainly being the tertiary alcohol, primary alconol and secondary alcohol) on by the FTA diaphragm of sample homogenization liquid wetting, make wetting FTA diaphragm only need 10 minutes can finish drying process at ambient temperature, shortened the time of sample nucleic acids for preparation greatly.When utilizing isothermal amplification method to detect cause of disease, generally adopt amplified reaction to finish the back at present and in reaction tubes, add nucleic acid dye to judge the method for detected result; Because the product amount of isothermal amplification is very big, thisly opens method that reaction tubes adds nucleic acid dye again and very easily cause follow-up sample to be checked contaminated and make detected result false positive occur; There are report and patent to adopt and in amplification reaction reagent, add the method decontamination risk of uracil dna glycosylase (UNG enzyme), but increased cost; The present invention adheres to nucleic acid dye the inside front or the lid inboard of amplified reaction pipe earlier, by shaking repeatedly up and down amplification reaction solution and nucleic acid dye are mixed again Deng after the amplified reaction end, do not open the amplified reaction pipe like this and just can finish the dyeing of amplified production, eliminated and opened the amplified reaction pipe and add in the nucleic acid dye process reaction solution again and may spill and cause the contaminated risk of follow-up sample to be checked.Be equipped with the FTA blocking in the standard step of sample nucleic acid, needing to use the special purified reagent of Whatman company (Britain) rinsing FTA diaphragm repeatedly; The present invention need not special purified reagent, adopts common distilled water rinsing can finish FTA diaphragm-operated purifying, has not only simplified the operation steps that detects, and has also reduced experimental cost.In order to make this detection method have stronger practicality in the production scene, the present invention assembles detecting required reagent and the equipment of TSV virus on the basis of the whole detection method of above-mentioned optimization, with it stdn, supportingization, has formed detection kit.
Prawn Taura syndrome field fast high-sensitive detecting kit of the present invention comprises:
(1) sampling tube is used for holding, grinds sample to be checked;
(2) rinsing tube, interior dress distilled water;
(3) nucleic acid denaturation pipe, interior dress TE damping fluid;
(4) augmentation detection pipe, interior dress amplification reaction solution and nucleic acid dye, the amplification reaction solution moiety is as follows: the upstream primer 1 of amplimer and downstream primer 1 each 1～2 μ M, the upstream primer 2 of amplimer and downstream primer 2 each 0.1～0.4 μ M, each 0.8～2.0mM of dATP, dTTP, dGTP and dCTP, MgCl
24～10mM, Betaine (trimethyl-glycine) 0.6～1.2M, Tris-HCl 10～40mM, KCl 10～20mM, MgSO
41～4mM, (NH
4)
2SO
46～12mM, TritonX-1000.05%～1.0%, ThermoScript II 1～20U, BstDNA polysaccharase 2～20U; Nucleic acid dye is a nucleic acid dye commonly used in the molecular biology research, includes but not limited to SYBR Green, GelRed, GelGreen, GoldView
TM, GeneFinder
TMDeng; And nucleic acid dye adheres to augmentation detection tube wall inside front or augmentation detection pipe lid inboard in advance;
(5) negative control pipe, interior dress do not have the FTA diaphragm of prawn Taura syndrome nucleic acid;
(6) positive control pipe, interior dress have adsorbed the FTA diaphragm of prawn Taura syndrome nucleic acid;
(7) rapid drying liquid is wetting ability and volatile alcohols material;
(8) FTA diaphragm, grinding rod, toothpick and suction pipe are packaged in the sterile bag respectively;
(9) packing box, in adorn a cystose or a sponge block that a plurality of apertures are arranged;
(10) working instructions.
Amplimer described in the above-mentioned TSV field fast high-sensitive detecting kit is according to TSV capsid protein gene conserved regions sequences Design, and its nucleotide sequence is as follows:
Upstream primer 1:5 '-CGGGCAAAGTAACCAAATCGCTTTTATGTTAACTAGACGTTCCAGT
Downstream primer 1:5 '-CCTTTGGCACAGATAATTCATTACGTTTTGCTACCATGAGTGAACCTA
Upstream primer 2:5 '-AACAGTTTGTCTCTCTCAGA
Downstream primer 2:5 '-TCGCCCTTATTCTTAGGAAT
Method with the above-mentioned detection kit detection of the present invention prawn TSV follows these steps to carry out:
(1) the sample thief prawn organizes about 0.02～1.0g to place sampling tube, with grinding rod sample prawn tissue is milled to pulpous state;
(2) it is fully wetting with the FTA diaphragm to dip in the sample prawn tissue of getting pulpous state with grinding rod;
(3) draw the rapid drying drop on above-mentioned FTA diaphragm, leave standstill 10～20min;
(4) with toothpick above-mentioned FTA diaphragm is transferred in the rinsing tube, concussion rinsing tube 3～5min is with washing FTA diaphragm;
(5) with toothpick the FTA diaphragm in the above-mentioned nucleic acid denaturation pipe is transferred in the augmentation detection pipe again, the augmentation detection pipe is placed under 55～65 ℃ of conditions be incubated 40～70min;
(6) with augmentation detection pipe whipping 1-3min repeatedly up and down, make amplification reaction solution and adhere to the abundant mixing of nucleic acid dye that augmentation detection tube wall or pipe cover;
(7) the downward whipping augmentation detection pipe of exerting oneself, make the amplification reaction solution that is mixed with nucleic acid dye in the pipe be collected in augmentation detection pipe bottom, with eyes observing response liquid, if reaction solution presents green then represents that the TSV detected result of this sample is positive,, reaction solution represents that the TSV detected result of this sample is negative if presenting orange-yellow.
In above-mentioned steps, since (4) step should with as the FTA diaphragm of the no prawn Taura syndrome nucleic acid of negative control, as the absorption of positive control the FTA diaphragm of prawn Taura syndrome nucleic acid, and the FTA diaphragm that has adsorbed sample nucleic acid handles together, until finishing detection reaction.
Positively effect of the present invention is: compile equipment such as reagent such as the required FTA diaphragm of TSV field quick detection, TE damping fluid, amplification reaction solution, nucleic acid dye and grinding rod, toothpick, suction pipe in test kit, make the detection of TSV to carry out rapidly and orderly, realized the sequencing and the stdn of testing process, make working specification, be difficult for makeing mistakes; The present invention can effectively detect institute's toxic strain of TSV according to the preferred designed amplimer of TSV capsid protein gene conserved regions sequence, and does not have homology with other viral capsid protein genes, and detection specificity is very good; FTA diaphragm after adopting alcohols material to sampling carries out rapid drying to be handled, and makes the preparation time of sample nucleic acid shorten greatly; Adopt built-in nucleic acid dye, need not open reaction tubes after reaction finishes can directly dye to reaction solution, avoid opening reaction tubes and added nucleic acid dye again and make follow-up sample to be checked be amplified product pollution and cause false-positive result, thereby improved this method reliability of applying in detection greatly.The present invention combines the reverse transcription of purpose nucleic acid (RNA) and amplification synchronously and carries out, and has saved the time, only needs just can finish in 1.5～2 hours the detection of prawn TSV; And testing process need not expensive plant and instrument such as whizzer, PCR instrument and gel imaging system, only needs water-bath or metal bath even simpler attemperator to get final product; And detected result is very easy to differentiate; Compare with the existing detection technique of prawn TSV, detection method cost of the present invention is very low, whole process does not relate to toxic reagent, all as safe as a house to operator and environment, and detection sensitivity is high, can substitute prawn Taura syndrome related detecting method such as pathological section method, electron microscopic observation method, antibody testing method and RT-PCR detection method before; Detection kit of the present invention not only can be used in the laboratory, also can use production scene in the open air, and is significant to epidemiological surveillance and the disease prevention and control of strengthening TSV, has highly application value.
Embodiment
The invention will be further described by the following examples.
Embodiment 1: prawn Taura syndrome field fast high-sensitive detecting kit
Test kit of the present invention is by form (packing that can detect 4 samples) with lower member:
(1) sampling tube, is used for holding, grinds sample to be checked by 4;
(2) rinsing tube, is equipped with the distilled water of 1ml by 6 in each pipe;
(3) nucleic acid denaturation pipe, 6, the TE damping fluid that 20 μ L are housed in each pipe (contains 10mM Tris-HCl and 1mMEDTA, pH8.0);
(4) augmentation detection pipe, 6, the nucleic acid dye of 25 μ L amplification reaction solutions and 1 μ L is housed in each pipe, the amplification reaction solution moiety is as follows: the upstream primer 1 of amplimer and downstream primer 1 each 1.6 μ M, the upstream primer 2 of amplimer and downstream primer 2 each 0.2 μ M, each 1.4mM of dATP, dTTP, dGTP and dCTP, MgCl
28mM, Betaine (trimethyl-glycine) 1.2M, Tris-HCl 20mM, KCl10mM, MgSO
42mM, (NH
4)
2SO
410mM, TritonX-1000.1%, ThermoScript II 5U, Bst archaeal dna polymerase 8U; The nucleic acid dye composition is the GeneFinder of 10 times of dilutions
TM, and nucleic acid dye has been adhered fixed in the pipe lid of augmentation detection pipe inboard.
(5) negative control pipe, 1, interior dress does not have the FTA diaphragm of prawn Taura syndrome nucleic acid;
(6) positive control pipe, 1, interior dress has adsorbed the FTA diaphragm of prawn Taura syndrome nucleic acid;
(7) rapid drying liquid, 1 the pipe, in adorn 500 μ L dehydrated alcohols;
(8) FTA diaphragm (4), grinding rod (4), toothpick (6) and suction pipe (1) are packaged in the sterile bag respectively;
(9) packing box (1), in adorn a sponge block that a plurality of apertures are arranged, 4 * 6 of apertures are arranged on the sponge block;
(10) working instructions, 1 part.
Embodiment 2 prawn Taura syndrome isothermal amplification detection methods of the present invention
Use the detection kit among the embodiment 1, follow these steps to carry out:
(1) the about 0.1g of sample thief prawn tissue places sampling tube, fast sample mill is broken to pulpous state with grinding rod;
(2) it is fully wetting with the FTA diaphragm to dip in the sample of getting pulpous state with grinding rod;
(3) draw the rapid drying drop on above-mentioned FTA diaphragm, with the gentle and quiet 10min that puts of FTA diaphragm chamber;
(4) with toothpick above-mentioned FTA diaphragm is transferred in the rinsing tube, rinsing tube concuss 3min;
(5) with toothpick the FTA diaphragm in the above-mentioned nucleic acid denaturation pipe is transferred in the augmentation detection pipe again, the augmentation detection pipe is placed under 57 ℃ of conditions be incubated 60min;
(6) with augmentation detection pipe whipping 2min repeatedly up and down, the abundant mixing of the nucleic acid dye that amplification reaction solution and pipe are covered;
(7) the downward whipping augmentation detection pipe of exerting oneself then, the amplification reaction solution that is mixed with nucleic acid dye in the pipe is gathered in augmentation detection pipe bottom, observe amplification reaction solution with eyes, if present green then represent that the TSV detected result of this sample is positive, if present orange-yellow then represent that the TSV detected result of this sample is negative.
In above-mentioned steps, go on foot since the 4th, the FTA diaphragm of no prawn Taura syndrome nucleic acid, the FTA diaphragm that adsorbed the FTA diaphragm of prawn Taura syndrome nucleic acid and adsorbed sample nucleic acid should be handled together, until finishing detection reaction, be used separately as the feminine gender and the positive control of detection.
FTA diaphragm described in the present invention is the FTA card that Britain Whatman company is used for preparing nucleic acid; With the FTA card cut into yardstick for long * wide be not less than 1 millimeter * 1 millimeter the scraps of paper or punch into diameter be not less than 1 millimeter the scraps of paper and promptly can be made into the FTA diaphragm.
Reagent used in the present invention and material: primer is synthetic by Shanghai biotechnology limited liability company; Disodium ethylene diamine tetraacetate, Tutofusin tris, dNTP, trimethyl-glycine (Betaine), dATP, dGTP, dCTP and dTTP, KCl, MgSO
4, (NH
4)
2SO
4, MgCl
2, TritonX-100 is available from Shanghai biotechnology limited liability company; The BstDNA polysaccharase that isothermal duplication is used, ThermoScript II etc. are available from NEB company; Nucleic acid dye GeneFinder
TMAvailable from Xiamen Baiweixin Biological Technology Co., Ltd..
＜110〉Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
＜120〉prawn Taura syndrome field fast high-sensitive detecting kit and detection method
<160>4
<210>1
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require to design according to prawn Taura syndrome capsid protein gene sequence and isothermal duplication design of primers, as the upstream primer 1 of prawn Taura syndrome isothermal duplication detection
<400>1
cgggcaaagt?aaccaaatcg?cttttatgtt?aactagacgt?tccagt?46
<210>2
<211>48
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require to design according to prawn Taura syndrome capsid protein gene sequence and isothermal duplication design of primers, as the downstream primer 1 of prawn Taura syndrome isothermal duplication detection
<400>2
cctttggcac?agataattca?ttacgttttg?ctaccatgag?tgaaccta?48
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require to design according to prawn Taura syndrome capsid protein gene sequence and isothermal duplication design of primers, as the upstream primer 2 of prawn Taura syndrome isothermal duplication detection
<400>3
aacagtttgt?ctctctcaga?20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉require to design according to prawn Taura syndrome capsid protein gene sequence and isothermal duplication design of primers, as the downstream primer 2 of prawn Taura syndrome isothermal duplication detection
<400>4
tcgcccttat?tcttaggaat?20
Claims (8)
1. prawn Taura syndrome field fast high-sensitive detecting kit is characterized in that this detection kit comprises:
(1) sampling tube is used for holding, grinds sample to be checked;
(2) rinsing tube, interior dress distilled water;
(3) nucleic acid denaturation pipe, interior dress TE damping fluid;
(4) augmentation detection pipe, interior dress amplification reaction solution and nucleic acid dye;
(5) negative control pipe, interior dress do not have the FTA diaphragm of prawn Taura syndrome nucleic acid;
(6) positive control pipe, interior dress have adsorbed the FTA diaphragm of prawn Taura syndrome nucleic acid;
(7) rapid drying liquid;
(8) FTA diaphragm, grinding rod, toothpick and suction pipe are packaged in the sterile bag respectively;
(9) packing box, in adorn a cystose or a sponge block that a plurality of apertures are arranged;
(10) working instructions.
2. detection kit as claimed in claim 1, wherein said nucleic acid dye is the nucleic acid dye of using in the molecular biology, includes but not limited to SYBR Green, GelRed, GelGreen, GoldViewT
MOr GeneFinder
TM
3. detection kit as claimed in claim 1 is characterized in that described rapid drying liquid is wetting ability and volatile alcohols material.
4. detection kit as claimed in claim 1 is characterized in that the nucleic acid dye in the above-mentioned augmentation detection pipe adheres to augmentation detection tube wall inside front or augmentation detection pipe pipe lid inboard.
5. detection kit as claimed in claim 1 is characterized in that described FTA diaphragm is that the scraps of paper that FTA card that Britain Whatman company is used for preparing nucleic acid cuts into 1 millimeter of yardstick form.
6. detection kit as claimed in claim 1, it is characterized in that above-mentioned amplification reaction solution is made up of following component: the upstream primer 1 of amplimer and downstream primer 1 each 1～2 μ M, the upstream primer 2 of amplimer and downstream primer 2 each 0.1～0.4 μ M, each 0.8～2.0mM of dATP, dTTP, dGTP and dCTP, MgCl
24～10mM, trimethyl-glycine 0.6～1.2M, Tris-HCl 10～40mM, KCl 10～20mM, MgSO
41～4mM, (NH
4)
2SO
46～12mM, Triton X-1000.05%～1.0%, ThermoScript II 1～20U has the active archaeal dna polymerase 2～20U of strand displacement.
7. detection kit as claimed in claim 6 is characterized in that the nucleotide sequence of amplimer is as follows in the amplification reaction solution:
Upstream primer 1:5 '-CGGGCAAAGTAACCAAATCGCTTTTATGTTAACTAGACGTTCCAGT
Downstream primer 1:5 '-CCTTTGGCACAGATAATTCATTACGTTTTGCTACCATGAGTGAACCTA
Upstream primer 2:5 '-AACAGTTTGTCTCTCTCAGA
Downstream primer 2:5 '-TCGCCCTTATTCTTAGGAAT
8. use the described detection kit of claim 1 to detect the method for prawn Taura syndrome, it is characterized in that this method comprises the following steps:
(1) the sample thief prawn organizes about 0.02～1.0g to place sampling tube, with grinding rod sample mill is broken to pulpous state;
(2) it is fully wetting with the FTA diaphragm to dip in the sample of getting pulpous state with grinding rod;
(3) draw the rapid drying drop on above-mentioned FTA diaphragm, leave standstill 10～20min;
(4) with toothpick above-mentioned FTA diaphragm is transferred in the rinsing tube, concussion rinsing tube 3～5min is with washing FTA diaphragm;
(5) with toothpick the FTA diaphragm in the above-mentioned nucleic acid denaturation pipe is transferred in the augmentation detection pipe again, the augmentation detection pipe is placed under 55～65 ℃ of conditions be incubated 40～70min;
(6) with augmentation detection pipe whipping 1-3min repeatedly up and down, make amplification reaction solution and adhere to the abundant mixing of nucleic acid dye that augmentation detection tube wall or pipe cover;
(7) downward whipping augmentation detection pipe, make the amplification reaction solution that is mixed with nucleic acid dye in the pipe be collected in augmentation detection pipe bottom, direct viewing amplification reaction solution color, if amplification reaction solution presents green then represents that the prawn Taura syndrome detected result of this sample is positive,, amplification reaction solution represents that the prawn Taura syndrome detected result of this sample is negative if presenting orange-yellow.
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| CN2009100178963A CN101643792B (en) | 2009-08-18 | 2009-08-18 | On-site rapid high-sensitivity detection kit and detection method of taura syndrome virus (TSV) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875980A (en) * | 2010-07-16 | 2010-11-03 | 中国水产科学研究院黄海水产研究所 | Kit and method for detecting Macrobrachium rosenbergii Nodavirus |
| CN101921872A (en) * | 2010-04-13 | 2010-12-22 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit and detection method of prawn yellow head virus |
| CN101921873A (en) * | 2010-04-13 | 2010-12-22 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and detection method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1199859A (en) * | 1997-05-20 | 1998-11-25 | 国家海洋局第三海洋研究所 | Fast detection method of white spot rhabdivirus of prawn |
| CN1114703C (en) * | 1998-03-24 | 2003-07-16 | 中国科学院上海生物化学研究所 | Polymerase chain-reaction test method for baculovirus of prawn |
| CN1118704C (en) * | 2000-12-08 | 2003-08-20 | 中国科学院水生生物研究所 | Reagent kit for diagnosing white-spot baculovirus of prawn and its test method |
| CN101372714B (en) * | 2008-09-07 | 2010-11-10 | 中国水产科学研究院黄海水产研究所 | Prawn white spot syndrome virus nucleic acid isothermal amplification detection reagent kit and detecting method |
-
2009
- 2009-08-18 CN CN2009100178963A patent/CN101643792B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921872A (en) * | 2010-04-13 | 2010-12-22 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit and detection method of prawn yellow head virus |
| CN101921873A (en) * | 2010-04-13 | 2010-12-22 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and detection method thereof |
| CN101921873B (en) * | 2010-04-13 | 2011-09-28 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit of prawn infectivity muscle necrosis virus and detection method thereof |
| CN101921872B (en) * | 2010-04-13 | 2011-09-28 | 中国水产科学研究院黄海水产研究所 | On-site rapid high-sensitivity detection kit and detection method of prawn yellow head virus |
| CN101875980A (en) * | 2010-07-16 | 2010-11-03 | 中国水产科学研究院黄海水产研究所 | Kit and method for detecting Macrobrachium rosenbergii Nodavirus |
| CN101875980B (en) * | 2010-07-16 | 2011-09-28 | 中国水产科学研究院黄海水产研究所 | Kit and method for detecting Macrobrachium rosenbergii Nodavirus |
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| CN101643792B (en) | 2011-02-09 |
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