CN1944666A - Primer sequence and detecting reagent kit for detecting prawn white spot syndrome virus - Google Patents
Primer sequence and detecting reagent kit for detecting prawn white spot syndrome virus Download PDFInfo
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- CN1944666A CN1944666A CN 200610016169 CN200610016169A CN1944666A CN 1944666 A CN1944666 A CN 1944666A CN 200610016169 CN200610016169 CN 200610016169 CN 200610016169 A CN200610016169 A CN 200610016169A CN 1944666 A CN1944666 A CN 1944666A
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Abstract
The present invention discloses primer sequence and detecting reagent kit for detecting prawn white sport syndrome virus. The detecting reagent kit contains the following reagents: mixture of hexadecyl trimethyl ammonium bromide, water, NaCl, EDTA and Tris-HCl; water solution of silica gel; water solution of ethaol; mixture of PCR buffer, Taq DNA polymerase and sterile double distilled water; mixture of nucleotide sequence of SEQ ID No. 1 and SEQ ID No. 2, dNTPs and MgCl2; genome total DNA of prawn white sport syndrome virus; sterile double distilled water; TBE electrophoretic buffer containing Tris-boric acid and EDTA; and agarose containing ethidium bromide. The present invention has primer sequence with optimized PCR condition and system, and establishes the PCR detecting method for detecting prawn white sport syndrome virus.
Description
Technical field
The present invention relates to the dna sequence dna in the biotechnology, the test kit that uses this sequence that a kind of virus is detected particularly relates to a kind of primer sequence and detection kit that is used to detect prawn ' s virus.
Background technology
Since the eighties in last century, China's water industry develops rapidly, and shared proportion rises year by year in the agriculture output value, has become one of four big mainstay industries of China's agricultural.But hydrocoles disease regrettably, especially due to illness the fulminant prevailing disease showed increased that causes of poison endangers very serious.In 1992, because spreading with popular of white spot syndrome virus (WSSV) caused the financial loss of more than one hundred million units for the aquatic products aquaculture.
The hydrocoles virus disease have latent period different in size, symptom is complicated and changeable, infectivity is strong and cause the characteristics of high mortality.Particularly virus is individual small, duplicate in host cell after infecting animal, and microbiotic is to the basic unrestraint effect of virus, and chemicals just may make host animal impaired or deadly before kill virus.Because the endotrophic characteristic of characteristic that hydrocoles is aquatic and virus, fundamentally eliminate intracellular virus and not influence host cell is impossible at present.Therefore must carry out the prevention work of hydrocoles virus disease, the basis of carrying out this work will be set up the quick diagnosis and the detection technique of virus exactly.
Research to the virus disease detection technique depends on virus disease pathology and STUDY ON PATHOGEN basis more, and the main method that adopts has research methods such as morphology, histopathology, immunology.Mainly comprise on-the-spot visual observation method, bioassay method, light microscopy, electron microscopy, immunofluorescence test procedure etc.These methods length consuming time, and to the dependence experience judge that this detection to virus has caused certain difficulty mostly.
Polymerase chain reaction (Polymerase chain reaction is called for short PCR) technology for detection is highly sensitive, high specificity, and round of visits is short, operates easy relatively.But still useless this kind method is to the report of white spot syndrome virus (WSSV) quick diagnosis and detection at present.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of primer sequence that is used to detect white spot syndrome virus (WSSV) is provided.
Second purpose of the present invention provides a kind of white spot syndrome virus (WSSV) to breed Penaeus vannamei, Chinese prawn, tigar prawn etc. and carries out tracking monitor, and has white spot syndrome virus (WSSV) detection kit easy, quick, sensitive, special characteristics.
Technical scheme of the present invention is summarized as follows:
A kind of primer sequence that is used to detect white spot syndrome virus (WSSV), it is made up of upstream primer and downstream primer, described upstream primer has the described nucleotide sequence of SEQ ID No1 in the sequence table, and described downstream primer has the described nucleotide sequence of SEQ ID No2.
A kind of white spot syndrome virus (WSSV) detection kit, form by following reagent:
Reagent A: the hexadecyl trimethyl ammonium bromide of 1-3g is added the water of 100ml, make and wherein contain 0.5-2.5M NaCl, 10-30mM ethylenediamine tetraacetic acid (EDTA) and 10-30mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make;
Reagent B: concentration is the aqueous solution of silica gel of 0.05g/mL-0.5g/mL;
Reagent C: concentration expressed in percentage by volume is the aqueous ethanolic solution of 60%-90%;
Reagent D: 10 times of PCR reaction buffer 2.5-5.0 μ L, Taq archaeal dna polymerase 1-2.5U, sterilization distilled water 12-18 μ L are mixed and made into;
Reagent E: with the described nucleotide sequence 0.2-1.0 of 10 μ M SEQ ID No1 μ L, the described nucleotide sequence 0.2-1.0 of 10 μ M SEQ ID No2 μ L, 10mM dNTPs 0.2-0.8 μ L, 25mM MgCl
21.5-3.5 μ L is mixed and made into;
Reagent F: genome total DNA of prawn white sport syndrome virus;
Reagent G: sterilization distilled water;
Reagent H: get 20 times of TBE electrophoretic buffers of 50mL, make and wherein contain 0.09mol hydroxymethyl aminomethane-boric acid, the 2mmol ethylenediamine tetraacetic acid (EDTA) is made;
Reagent I:0.3-0.6g agarose makes wherein to contain ethidium bromide 15-30 μ g.
A kind of white spot syndrome virus (WSSV) detection kit, preferred scheme is:
Reagent A is: the hexadecyl trimethyl ammonium bromide of 2g is added the water of 100ml, make and wherein contain 1.4M NaCl, 20mM ethylenediamine tetraacetic acid (EDTA) and 20mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make;
Reagent B is: concentration is the aqueous solution of silica gel of 0.3g/mL;
Reagent C is: concentration expressed in percentage by volume is 75% aqueous ethanolic solution;
Reagent D is: 10 times of PCR reaction buffers, 2.5 μ L, Taq archaeal dna polymerase 1.25U, sterilization distilled water 16.75 μ L are mixed and made into;
Reagent E is: with the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No1, and the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No2,10mM dNTPs 0.5 μ L, 25mM MgCl
22.5 μ L is mixed and made into;
Reagent F: genome total DNA of prawn white sport syndrome virus;
Reagent G: sterilization distilled water;
Reagent H: get 20 times of TBE electrophoretic buffers of 50mL, make and wherein contain 0.09mol hydroxymethyl aminomethane-boric acid, the 2mmol ethylenediamine tetraacetic acid (EDTA) is made;
Reagent I is: the 0.3g agarose makes wherein to contain ethidium bromide 20 μ g.
Genome total DNA of prawn white sport syndrome virus is extracted with following method: the gill filament 50-100mg that gets the prawn that infects white spot syndrome virus (WSSV) with the sterilization scissors, place in the 1.5mlEppendorf pipe of sterilization, add 150 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, fully shred tissue, add 700 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, 25 ℃ of effect 2h, add 600 μ L volume ratios and be phenol/chloroform/primary isoamyl alcohol of 25: 24: 1, mixing 30s, through the centrifugal 5min of 12000r/m, get the upper strata water, add 700 μ L volume ratios and be chloroform/primary isoamyl alcohol of 24: 1, mixing 30s, the centrifugal 5min of 12000r/m gets the upper strata water, adds-20 ℃ of dehydrated alcohols of two volumes, precipitate 8-12 hour, the centrifugal 30min of 15000r/m discards supernatant liquor, dries, the dissolving of 100ul sterilization distilled water ,-20 ℃ of preservations.
Advantage of the present invention:
Utilize the gene conserved sequence of white spot syndrome virus (WSSV) to design a pair of primer, optimize PCR reaction conditions and reaction system, set up the PCR detection method that detects white spot syndrome virus (WSSV), assemble out easy, quick, sensitive, special a kind of white spot syndrome virus (WSSV) detection kit on this basis, this test kit can be used for the tracking monitor to the white spot syndrome virus (WSSV) of culturing Penaeus vannamei, Chinese prawn, tigar prawn etc., has very high practical value.
Description of drawings
Fig. 1 is for detecting white spot syndrome virus (WSSV) electrophoresis result figure.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
A kind of primer sequence that is used to detect white spot syndrome virus (WSSV), be that it is made up of upstream primer and downstream primer, described upstream primer has the described nucleotide sequence of SEQ ID No1 in the sequence table, and described downstream primer has the described nucleotide sequence of SEQ ID No2.
Sequence table
SEQ?ID?No1：
5’--CCAAGACATACTAGCGGATA--3’
SEQ?ID?No2：
5’--GACGACCCTGACAGAATTAC--3’
Embodiment 2
A kind of white spot syndrome virus (WSSV) detection kit, form by following reagent:
Reagent A is: the hexadecyl trimethyl ammonium bromide of 2g is added the water of 100ml, make and wherein contain 1.4M NaCl, 20mM ethylenediamine tetraacetic acid (EDTA) (EDTA) and 20mM hydroxymethyl aminomethane-hydrochloric acid (Tris-HCl), adjusting pH=7.5 makes;
Reagent B is: concentration is the aqueous solution of silica gel of 0.3g/mL;
Reagent C is: concentration expressed in percentage by volume is 75% aqueous ethanolic solution; (reagent A, reagent B and reagent C are white spot syndrome virus (WSSV) extracting genome DNA reagent)
Reagent D (PCR reaction premix reagent): 10 times of PCR reaction buffers, 2.5 μ L, Taq archaeal dna polymerase 1.25U, sterilization distilled water 16.75 μ L are mixed and made into;
Reagent E (PCR reaction primer reagent): with the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No1, the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No2,10mM dNTPs 0.5 μ L, 25mM MgCl
22.5 μ L is mixed and made into;
Reagent F (positive control): genome total DNA of prawn white sport syndrome virus; Genome total DNA of prawn white sport syndrome virus is extracted with following method: the gill filament 100mg that gets the prawn that infects white spot syndrome virus (WSSV) with the sterilization scissors, place in the 1.5mlEppendorf pipe of sterilization, add 150 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, fully shred tissue, add 700 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, 25 ℃ of effect 2h, add 600 μ L volume ratios and be phenol/chloroform/primary isoamyl alcohol of 25: 24: 1, mixing 30s, through the centrifugal 5min of 12000r/m, get the upper strata water, add 700 μ L volume ratios and be chloroform/primary isoamyl alcohol of 24: 1, mixing 30s, the centrifugal 5min of 12000r/m gets the upper strata water, adds-20 ℃ of dehydrated alcohols of two volumes, precipitate 10 hours, the centrifugal 30min of 15000r/m discards supernatant liquor, dries, the dissolving of 100ul sterilization distilled water ,-20 ℃ of preservations.
Reagent G (blank): sterilization distilled water; (redistilled water)
Reagent H (electrophoresis reagent): get 20 times of TBE electrophoretic buffers of 50mL, make and wherein contain 0.09mol hydroxymethyl aminomethane-boric acid (Tris-boric acid), the 2mmol ethylenediamine tetraacetic acid (EDTA) is made (in this ratio, can obtain the reagent H of any volume);
Reagent I (colouring reagents): the 0.3g agarose makes wherein to contain ethidium bromide 20 μ g.
Sterilization toothpick one bag.
Embodiment 3
A kind of step of detection white spot syndrome virus (WSSV) of white spot syndrome virus (WSSV) detection kit:
1, gets the about 50-100mg of sample tissue to be checked under the sterile state, add 200 μ L reagent A, smash 1-2min to pieces with the sterilization toothpick;
2, the centrifugal 3min of 12000rpm/min gets supernatant, adds 20 μ L reagent B (shaking up with preceding need), and room temperature is placed 3-5min, constantly shakes up therebetween;
3, the centrifugal 30sec of 10000rpm/min discards supernatant liquor, adds 200 μ L reagent C, and the centrifugal 30sec of 10000rpm/min discards supernatant liquor;
4, open the centrifuge tube lid, place 60 ℃ of 3min, add 20 μ L reagent G suspension precipitation, centrifugal slightly, get the template of supernatant as the PCR reaction;
5, pcr amplification: get 19 μ L reagent D and mix with 4 μ L reagent E, add 2 μ L PCR reaction template (with 20 μ L reagent G dissolved reagent F), get 2 μ L reagent F and reagent G simultaneously and add respectively in two other 19 μ L reagent D and the 4 μ L reagent E blended PCR reaction tubess, respectively as positive control and blank, on the PCR instrument, increase, reaction conditions is: 94 ℃ of pre-sex change of 4min, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 29 circulations, 72 ℃ of 10min, 4 ℃ of preservations;
6, after reaction finishes, get reagent H and do 40 times of dilutions with distilled water.Reagent I is poured in the triangular flask of 100mL, add the reagent H after diluting, heating, limpid until solution, the glue groove is poured in cooling into slightly;
7, get 5-10 μ L PCR reaction product and add 2 μ L sample-loading buffers, behind 1% agarose gel electrophoresis 30-35min, on ultraviolet device, observe.If occur reacting band at the 235bp place, then be the white spot syndrome virus (WSSV) positive, if no band is negative, the reaction band should appear at the 235bp place in positive mark, the blank reactionless band (see Fig. 1,1 is the 235bp place among the figure) of answering.
The PCR method that this test kit is set up can detect white spot syndrome virus (WSSV) from pathological material of disease in 5-6h.
The present invention relates generally to the cause of disease white spot syndrome virus (WSSV) (white spot sydrome virus be called for short WSSV) of object for culturing Penaeus vannamei, Chinese prawn, tigar prawn etc.White spot syndrome virus (WSSV) is to adopt baculovirus of prawn polymerase chain reaction (PCR) detection method in People's Republic of China's inspection and quarantining for import/export industry standard of quality supervision and test quarantine general bureau of People's Republic of China (PRC) issue to detect.
Embodiment 4
A kind of white spot syndrome virus (WSSV) detection kit, form by following reagent:
Reagent A: the hexadecyl trimethyl ammonium bromide of 1g is added the water of 100ml, make and wherein contain 0.5M NaCl, 10mM ethylenediamine tetraacetic acid (EDTA) and 10mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make;
Reagent B: concentration is the aqueous solution of silica gel of 0.05g/mL;
Reagent C: concentration expressed in percentage by volume is 90% aqueous ethanolic solution;
Reagent D: 10 times of PCR reaction buffers, 2.5 μ L, Taq archaeal dna polymerase 1U, sterilization distilled water 12 μ L are mixed and made into;
Reagent E: with the described nucleotide sequence 0.2 μ L of 10 μ M SEQ ID No1, the described nucleotide sequence 0.2 μ L of 10 μ M SEQ ID No2,10mM dNTPs 0.2 μ L, 25mM MgCl
21.5 μ L is mixed and made into;
Reagent F: genome total DNA of prawn white sport syndrome virus;
Reagent G: sterilization distilled water;
Reagent H: get 20 times of TBE electrophoretic buffers of 50mL, make and wherein contain 0.09mol hydroxymethyl aminomethane-boric acid, the 2mmol ethylenediamine tetraacetic acid (EDTA) is made;
Reagent I:0.3 agarose makes wherein to contain ethidium bromide 15 μ g.
Embodiment 5
A kind of white spot syndrome virus (WSSV) detection kit, form by following reagent:
Reagent A: the hexadecyl trimethyl ammonium bromide of 3g is added the water of 100ml, make and wherein contain 2.5M NaCl, 30mM ethylenediamine tetraacetic acid (EDTA) and 30mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make;
Reagent B: concentration is the aqueous solution of silica gel of 0.05g/mL;
Reagent C: concentration expressed in percentage by volume is 60% aqueous ethanolic solution;
Reagent D: 10 times of PCR reaction buffers, 5.0 μ L, Taq archaeal dna polymerase 2.5U, sterilization distilled water 18 μ L are mixed and made into;
Reagent E: with the described nucleotide sequence 1.0 μ L of 10 μ M SEQ ID No1, the described nucleotide sequence 1.0 μ L of 10 μ M SEQ ID No2,10mM dNTPs 0.8 μ L, 25mM MgCl
23.5 μ L is mixed and made into;
Reagent F: genome total DNA of prawn white sport syndrome virus;
Reagent G: sterilization distilled water;
Reagent H: get 20 times of TBE electrophoretic buffers of 100mL, make and wherein contain 0.18mol hydroxymethyl aminomethane-boric acid, the 4mmol ethylenediamine tetraacetic acid (EDTA) is made;
Reagent I:0.6g agarose makes wherein to contain ethidium bromide 30 μ g.
Claims (4)
1. primer sequence that is used to detect white spot syndrome virus (WSSV), it is characterized in that it is made up of upstream primer and downstream primer, described upstream primer has the described nucleotide sequence of SEQ ID No1 in the sequence table, and described downstream primer has the described nucleotide sequence of SEQ ID No2 in the sequence table.
2. white spot syndrome virus (WSSV) detection kit is characterized in that being made up of following reagent:
Reagent A: the hexadecyl trimethyl ammonium bromide of 1-3g is added the water of 100ml, make and wherein contain 0.5-2.5M NaCl, 10-30mM ethylenediamine tetraacetic acid (EDTA) and 10-30mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make;
Reagent B: concentration is the aqueous solution of silica gel of 0.05g/mL-0.5g/mL;
Reagent C: concentration expressed in percentage by volume is the aqueous ethanolic solution of 60%-90%;
Reagent D: 10 times of PCR reaction buffer 2.5-5.0 μ L, Taq archaeal dna polymerase 1-2.5U, sterilization distilled water 12-18 μ L are mixed and made into;
Reagent E: with the described nucleotide sequence 0.2-1.0 of 10 μ M SEQ ID No1 μ L, the described nucleotide sequence 0.2-1.0 of 10 μ M SEQ ID No2 μ L, 10mM dNTPs 0.2-0.8 μ L, 25mM MgCl
21.5-3.5 μ L is mixed and made into;
Reagent F: genome total DNA of prawn white sport syndrome virus;
Reagent G: sterilization distilled water;
Reagent H: get 20 times of TBE electrophoretic buffers of 50mL, make and wherein contain 0.09mol hydroxymethyl aminomethane-boric acid, the 2mmol ethylenediamine tetraacetic acid (EDTA) is made;
Reagent I:0.3-0.6g agarose makes wherein to contain ethidium bromide 15-30 μ g.
3. a kind of white spot syndrome virus (WSSV) detection kit according to claim 2, it is characterized in that described reagent A is: the water that the hexadecyl trimethyl ammonium bromide of 2g is added 100ml, make and wherein contain 1.4M NaCl, 20mM ethylenediamine tetraacetic acid (EDTA) and 20mM hydroxymethyl aminomethane-hydrochloric acid, regulate pH=7.5 and make; Described reagent B is: concentration is the aqueous solution of silica gel of 0.3g/mL; Described reagent C is: concentration expressed in percentage by volume is 75% aqueous ethanolic solution; Described reagent D is: 10 times of PCR reaction buffers, 2.5 μ L, Taq archaeal dna polymerase 1.25U, sterilization distilled water 16.75 μ L are mixed and made into; Described reagent E is: with the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No1, and the described nucleotide sequence 0.5 μ L of 10 μ M SEQ ID No2,10mM dNTPs 0.5 μ L, 25mM MgCl
22.5 μ L is mixed and made into; Described reagent I is: the 0.3g agarose makes wherein to contain ethidium bromide 20 μ g.
4. according to claim 2 or 3 described a kind of white spot syndrome virus (WSSV) detection kit, it is characterized in that described genome total DNA of prawn white sport syndrome virus extracts with following method: the gill filament 50-100mg that gets the prawn that infects white spot syndrome virus (WSSV) with the sterilization scissors, place in the 1.5ml Eppendorf pipe of sterilization, add 150 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, fully shred tissue, add 700 μ L mass percents and be 1% the hexadecyl trimethyl ammonium bromide aqueous solution, 25 ℃ of effect 2h, add 600 μ L volume ratios and be phenol/chloroform/primary isoamyl alcohol of 25: 24: 1, mixing 30s, through the centrifugal 5min of 12000r/m, get the upper strata water, add 700 μ L volume ratios and be chloroform/primary isoamyl alcohol of 24: 1, mixing 30s, the centrifugal 5min of 12000r/m, get the upper strata water,-20 ℃ of dehydrated alcohols that add two volumes,-20 ℃ precipitate 8-12 hour, the centrifugal 30min of 15000r/m, discard supernatant liquor, dry the dissolving of 100ul sterilization distilled water ,-20 ℃ of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921741B (en) * | 2009-11-30 | 2012-06-06 | 浙江大学 | Relative protein for resisting against prawn white spot syndrome viruses (WSSV), characteristic sequence and application thereof |
| CN102952902A (en) * | 2012-11-27 | 2013-03-06 | 天津市水生动物疫病预防控制中心 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit for shrimp white spot syndrome virus |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1188531C (en) * | 2003-05-06 | 2005-02-09 | 中山大学 | Prawn white spot complex virogene diagnostic kit and detecting method thereof |
| CN1197977C (en) * | 2003-05-19 | 2005-04-20 | 中国科学院武汉病毒研究所 | Method for hybrid detecting nucleic acid complex point of shrimp viral |
| CN1243981C (en) * | 2003-07-30 | 2006-03-01 | 中国科学院武汉病毒研究所 | Method for detecting prawn virus by compound polymerase chain -enzyme-linked immune reaction |
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- 2006-10-19 CN CNB2006100161691A patent/CN100402668C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921741B (en) * | 2009-11-30 | 2012-06-06 | 浙江大学 | Relative protein for resisting against prawn white spot syndrome viruses (WSSV), characteristic sequence and application thereof |
| CN102952902A (en) * | 2012-11-27 | 2013-03-06 | 天津市水生动物疫病预防控制中心 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit for shrimp white spot syndrome virus |
| CN102952902B (en) * | 2012-11-27 | 2014-04-02 | 天津市水生动物疫病预防控制中心 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit for shrimp white spot syndrome virus |
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