CN1687447A - Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof - Google Patents
Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof Download PDFInfo
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Abstract
This invention provides an apparatus which simultaneously detects the fish virus of IHNV, IPNV, and VHSV. It also discloses the method of detecting by this apparatus of solution box. It character in that it's fast and simple with high sensitivity to detect the three bacteriums qualitative. This invention can be used to detect three kinds of disease of import and export, and to monitor epidemic situation of breeding farm to avoid disease dissemination, so that it has a high value.
Description
Technical field
The present invention relates to the particularly detection test kit of fish virus of a kind of hydrocoles, the test kit of particularly a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus the invention still further relates to the detection method of aforementioned synchronous detection.
Background technology
Viral haemorrhagic septicaemia virus (VHSV), infectious pancreatic necrosis virus (IPNV) and fish infectious hematopoietic necrosis virus (IHNV) are with the two class transmissible diseases that are listed in " People's Republic of China (PRC) enter the territory animal one, two class transmissible diseases, parasitosis register ", VHS and IHN are respectively two classes and the three class eqpidemic diseases of " People's Republic of China's animal epidemic prevention method " regulation, and these three kinds is the disease in the OIE of the International Office of Epizootics register.IHN and VHS virus all belong to the Rhabdoviridae single strand RNA virus, IHN virus causes the fatal disease of the multiple fish of salmon section, by the propagation such as water, feed and ight soil of polluting, juvenile fish and fry mortality ratio can reach 100%, adult fish infects can be with poison and the poison that looses all the life, and VHS virus can cause the multiple fish morbidity of the salmon section at various ages, and mortality ratio reaches 80-100%, IPN Tobamovirus birnavirus section diplornavirus causes the acute hyperinfection disease of a lot of hydrocoles that comprise the salmon flying fish.These 3 kinds of virus diseases all are the acute hyperinfection diseases that caused by RNA viruses, can encroach on multiple seawater and cultured freshwater fish, popular in Europe, the United States, Japan and Korea S. and part Asian countries, can by the fish body and lay eggs, propagation such as feed of seminal fluid, urine, ight soil and pollution, water, plant is caused havoc, endanger very serious.To the best control method of above-mentioned 3 diseases is to strengthen plant is detected, and prevents that virus from importing the shoal of fish into.At present also not to these three kinds of test kits that fish disease detects, at present the detection method that three kinds of fish diseases are generally adopted is to inoculate responsive fish and cell carries out virus multiplication with pathological material of disease, separation and purification virus, carrying out virus with serum neutralization, immunofluorescence and enzyme-linked immune detection method then identifies, its operation steps complexity is loaded down with trivial details, for up to 2-4 week.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, the test kit of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus is provided, can carries out quick and easy, detection rapidly synchronously to above-mentioned 3 kinds of viruses with this test kit.
The invention also discloses a kind of detection method of using mentioned reagent box synchronous detection infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is the test kit of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus, is characterized in that it is made of following article,
(1) negative control does not contain the tissue extract of IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV, 1;
(2) positive control contains the solution of IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV, 1;
(3) dNTP, 1;
(4) contain Mg
2+The PCR damping fluid, 1;
(5) primer is to ihnf/ihnr, each 1;
Ihnf is: 5 '-gttaacttcaacgccaacagg,
Ihnr is: 5 '-tgaagtacccctccccgagcatcc;
(6) primer is to ipnf/ipnr, each 1;
Ipnf is: 5 '-ccgcaacttacttgagatccattatgc,
Ipnr is: 5 '-cgtctggttcagattccacctgtagtg;
(7) primer is to vhsf/vhsr, each 1;
Vhsf is: 5 '-cgaccagctcaactcaggtgtcc,
Vhsr is: 5 '-ccaggtcggtcttgatccattctgtc;
(8) archaeal dna polymerase, 1;
(9) reversed transcriptive enzyme, 1;
(10) RNA enzyme inhibitors, 1;
(11) 100%DEPC solution, 1;
(12) the packing box is 1,1 of cystose, and its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that some quantity are arranged on the cystose is respectively applied for and places above-mentioned article.
Technical problem to be solved by this invention can also further realize by following technical scheme.The test kit of above-described a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus is characterized in that it can also contain following article:
(1) RNA extracts and uses Trizol liquid, 1;
(2) chloroform, 1;
(3) primary isoamyl alcohol, 1;
(4) 70% ethanol, 1;
(5) contain the aqueous solution of 0.01%DEPC, 1.
Technical problem to be solved by this invention can also further realize by following technical scheme.The present invention is the detection method of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus, is characterized in, adopts above-mentioned test kit, and its step is as follows,
(1) extraction of RNA template in the sample: get susceptible tissue sample homogenate or suspected virus and infect suspension 100-500 μ l, add 500-800 μ l Trizol reagent, vortex 30s, add chloroform and the primary isoamyl alcohol mixed solution of 200 μ l in ratio preparation in 24: 1, vortex 30s, the centrifugal 5-3min of 10000-15000r/min, get supernatant, it is the same centrifugal to add the equivalent aqueous isopropanol, gets precipitation, add 70% ethanolic soln of 300 μ l the same centrifugal after, remove supernatant, air-dry, add the 0.01%DEPC aqueous solution of 50 μ l, get 2-10 μ l and be used for RT-PCR;
(2) preparation of RT-PCR premix: elder generation adds each reagent in advance by following art formula and mixes, and needs the centrifugal several seconds before each reagent adds,
dNTP 1μl；
Contain Mg
2+The PCR damping fluid, 5 μ l;
Primer is to ihnf/ihnr 2 μ l;
Primer is to ipnf/ipnr, 2 μ l;
Primer is to vhsf/vhsr, 2 μ l;
Archaeal dna polymerase, 0.5 μ l;
Reversed transcriptive enzyme, 0.5 μ l;
The RNA enzyme inhibitors, 0.25 μ l;
The 0.01%DEPC aqueous solution, some;
In above-mentioned mixed solution, add respectively then:
The RNA template, 2-10 μ l gets RT-PCR premix A, cumulative volume 50 μ l;
Negative control, 5 μ l get RT-PCR premix B, cumulative volume 50 μ l;
Positive control, 5 μ l get RT-PCR premix C, cumulative volume 50 μ l;
(3) above-mentioned RT-PCR premix A, B, C are packed into PCR reaction tubes places gene-amplificative instrament, does not have the heat lid as instrument, adds the capping of 1-2 dropstone wax oil, and it is as follows that the RT-PCR loop parameter is set, and increases:
42℃×30min
95℃×4min
72℃×10min
25 ℃ * 1min or end
(4) after amplified reaction finishes, get 10-15 μ l and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 2% agarose gel electrophoresis, voltage is observed down in uv analyzer after pressing 5V/cm electrophoresis 30-40min, and negative control and positive control are correct, sample RNA extracting solution is if band occurs at 625bp, 371bp, 206bp place, then be respectively VHSV, IHNV and the IPNV positive, illustrate and carry three kinds of viruses in the testing sample, if one of them band occurs, then Dui Ying viral detected result is positive, otherwise negative.
Test kit of the present invention both can be used to detect simultaneously three kinds of viruses, also can be used to detect separately wherein each virus, when detecting separately, only right with the Auele Specific Primer of a kind of virus wherein, during as independent detection IHNV, add the ihnf/ihnr primer, do not add vhsf/vhsr and ipnf/ipnr primer, other reagent are constant, by that analogy.
Compared with prior art, use test kit of the present invention and detection method and can carry out qualitative detection IHNV, IPNV and three kinds of viruses of VHSV, not only can be used for carrying out IHNV, IPNV and the various single viral single tube RT-PCR of VHSV are detected, also can be used for 3 kinds of viral single tube RT-PCR are detected simultaneously and rapidly.Beginning whole process from sample preparation during detection can be controlled within 5 hours.Detection method of the present invention has avoided conventional elder generation that the RNA sample is carried out reverse transcription RT one-tenth cDNA, the reverse transcription product that takes a morsel again carries out two step complex operations steps and times, the reagent consumption of pcr amplification, can carry out qualitative detection to three kinds of viruses, easy to be quick, specificity is good, and is highly sensitive, can be used for the pass in and out detection of 3 kinds of fish diseases of fish and the epidemic monitoring of plant, anti-system eqpidemic disease spread and epidemic has very high practical value.Can be applicable to export aquarium fish, fish and products thereof monitoring quarantine, China hydrocoles plant is carried out eqpidemic disease diagnosis, the investigation of epidemic situation, and the detection of import hydrocoles and product.The inventive method accurately, reliable, fast, simple, easy to operate, be convenient to promote.
Description of drawings
Accompanying drawing is RT-PCR amplification figure of the present invention.Swimming lane 1:DNA mark, from top to bottom: 1116,883,692,500,400,331,242,190,110bp; Swimming lane 2/3,5/6: the positive control that contains VHSV, IHNV and IPNV; Swimming lane 4/7: negative control.
Embodiment
With reference to the accompanying drawings, further describe concrete technology implementation scheme of the present invention.
Embodiment 1.The test kit of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus, it is made of following article,
(1) negative control does not contain the tissue extract of IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV, 1;
(2) positive control contains the solution of IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV, 1;
(3) dNTP, 1;
(4) contain Mg
2+The PCR damping fluid, 1;
(5) primer is to ihnf/ihnr, each 1;
Ihnf is: 5 '-gttaacttcaacgccaacagg,
Ihnr is: 5 '-tgaagtacccctccccgagcatcc;
(6) primer is to ipnf/ipnr, each 1;
Ipnf is: 5 '-ccgcaacttacttgagatccattatgc,
Ipnr is: 5 '-cgtctggttcagattccacctgtagtg;
(7) primer is to vhsf/vhsr, each 1;
Vhsf is: 5 '-cgaccagctcaactcaggtgtcc,
Vhsr is: 5 '-ccaggtcggtcttgatccattctgtc;
(8) archaeal dna polymerase, 1;
(9) reversed transcriptive enzyme, 1;
(10) RNA enzyme inhibitors, 1;
(11) 100%DEPC solution, 1;
(12) the packing box is 1,1 of cystose, and its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that some quantity are arranged on the cystose is respectively applied for and places above-mentioned article.
Embodiment 2.Test kit among the embodiment 1, it also contains following article:
(1) RNA extracts and uses Trizol liquid, 1;
(2) chloroform, 1;
(3) primary isoamyl alcohol, 1;
(4) 70% ethanol, 1;
(5) contain the aqueous solution of 0.01%DEPC, 1.
Embodiment 3.With reference to accompanying drawing.The detection method of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus adopts embodiment 1 described test kit, and all the other required reagent are provided for oneself by the tester, and its step is as follows,
(1) extraction of RNA template in the sample: get susceptible tissue sample homogenate or suspected virus and infect suspension 100 μ l, add 500 μ l Trizol reagent, vortex 30s, add chloroform and the primary isoamyl alcohol mixed solution of 200 μ l in ratio preparation in 24: 1, vortex 30s, the centrifugal 5min of 10000r/min, get supernatant, it is the same centrifugal to add the equivalent aqueous isopropanol, gets precipitation, add 70% ethanolic soln of 300 μ l the same centrifugal after, remove supernatant, air-dry, add the 0.01%DEPC aqueous solution of 50 μ l, get 2 μ l and be used for RT-PCR;
(2) preparation of RT-PCR premix: elder generation adds each reagent in advance by following art formula and mixes, and needs the centrifugal several seconds before each reagent adds,
dNTP 1μl；
Contain Mg
2+The PCR damping fluid, 5 μ l;
Primer is to ihnf/ihnr 2 μ l;
Primer is to ipnf/ipnr, 2 μ l;
Primer is to vhsf/vhsr, 2 μ l;
Archaeal dna polymerase, 0.5 μ l;
Reversed transcriptive enzyme, 0.5 μ l;
The RNA enzyme inhibitors, 0.25 μ l;
The 0.01%DEPC aqueous solution, some;
In above-mentioned mixed solution, add respectively then:
The RNA template, 2 μ l get RT-PCR premix A, cumulative volume 50 μ l;
Negative control, 5 μ l get RT-PCR premix B, cumulative volume 50 μ l;
Positive control, 5 μ l get RT-PCR premix C, cumulative volume 50 μ l;
(3) above-mentioned RT-PCR premix A, B, C are packed into PCR reaction tubes places gene-amplificative instrament, does not have the heat lid as instrument, adds the capping of 1-2 dropstone wax oil, and it is as follows that the RT-PCR loop parameter is set, and increases:
42℃×30min
95℃×4min
72℃×10min
25℃×1min
(4) after amplified reaction finishes, get 10 μ l and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 2% agarose gel electrophoresis, voltage is observed down in uv analyzer after pressing 5V/cm electrophoresis 30min, and negative control and positive control are correct, sample RNA extracting solution is if band occurs at 625bp, 371bp, 206bp place, then be respectively VHSV, IHNV and the IPNV positive, illustrate and carry three kinds of viruses in the testing sample, if one of them band occurs, then Dui Ying viral detected result is positive, otherwise negative.
Embodiment 4.With reference to accompanying drawing.The detection method of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus adopts embodiment 2 described test kits, and its step is as follows,
(1) extraction of RNA template in the sample: get susceptible tissue sample homogenate or suspected virus and infect suspension 500 μ l, add 800 μ l Trizol reagent, vortex 30s, add chloroform and the primary isoamyl alcohol mixed solution of 200 μ l in ratio preparation in 24: 1, vortex 30s, the centrifugal 3min of 15000r/min, get supernatant, it is the same centrifugal to add the equivalent aqueous isopropanol, gets precipitation, add 70% ethanolic soln of 300 μ l the same centrifugal after, remove supernatant, air-dry, add the 0.01%DEPC aqueous solution of 50 μ l, get 10 μ l and be used for RT-PCR;
(2) preparation of RT-PCR premix: elder generation adds each reagent in advance by following art formula and mixes, and needs the centrifugal several seconds before each reagent adds,
dNTP 1μl；
Contain Mg
2+The PCR damping fluid, 5 μ l;
Primer is to ihnf/ihnr 2 μ l;
Primer is to ipnf/ipnr, 2 μ l;
Primer is to vhsf/vhsr, 2 μ l;
Archaeal dna polymerase, 0.5 μ l;
Reversed transcriptive enzyme, 0.5 μ l;
The RNA enzyme inhibitors, 0.25 μ l;
The 0.01%DEPC aqueous solution, some;
In above-mentioned mixed solution, add respectively then:
The RNA template, 10 μ l get RT-PCR premix A, cumulative volume 50 μ l;
Negative control, 5 μ l get RT-PCR premix B, cumulative volume 50 μ l;
Positive control, 5 μ l get RT-PCR premix C, cumulative volume 50 μ l;
(3) above-mentioned RT-PCR premix A, B, C are packed into PCR reaction tubes places gene-amplificative instrament, does not have the heat lid as instrument, adds the capping of 1-2 dropstone wax oil, and it is as follows that the RT-PCR loop parameter is set, and increases:
42℃×30min
95℃×4min
72℃×10min
(4) after amplified reaction finishes, get 15 μ l and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 2% agarose gel electrophoresis, voltage is observed down in uv analyzer after pressing 5V/cm electrophoresis 40min, and negative control and positive control are correct, sample RNA extracting solution is if band occurs at 625bp, 371bp, 206bp place, then be respectively VHSV, IHNV and the IPNV positive, illustrate and carry three kinds of viruses in the testing sample, if one of them band occurs, then Dui Ying viral detected result is positive, otherwise negative.
Claims (3)
1, the test kit of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus is characterized in that it is made of following article,
(1) negative control does not contain IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV
Tissue extract, 1;
(2) positive control contains IHNV, IPNV and 3 kinds of viral nucleic acid compositions of VHSV
Solution, 1;
(3) dNTP, 1;
(4) contain Mg
2+The PCR damping fluid, 1;
(5) primer is to ihnf/ihnr, each 1;
Ihnf is: 5 '-gttaacttcaacgccaacagg,
Ihnr is: 5 '-tgaagtacccctccccgagcatcc;
(6) primer is to ipnf/ipnr, each 1;
Ipnf is: 5 '-ccgcaacttacttgagatccattatgc,
Ipnr is: 5 '-cgtctggttcagattccacctgtagtg;
(7) primer is to vhsf/vhsr, each 1;
Vhsf is: 5 '-cgaccagctcaactcaggtgtcc,
Vhsr is: 5 '-ccaggtcggtcttgatccattctgtc;
(8) archaeal dna polymerase, 1;
(9) reversed transcriptive enzyme, 1;
(10) RNA enzyme inhibitors, 1;
(11) 100%DEPC solution, 1;
(12) the packing box is 1,1 of cystose, and its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that some quantity are arranged on the cystose is respectively applied for and places above-mentioned article.
2, the test kit of a kind of synchronous detection fish infectious hematopoietic necrosis virus according to claim 1, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus is characterized in that it can also contain following article:
(1) RNA extracts and uses Trizol liquid, 1;
(2) chloroform, 1;
(3) primary isoamyl alcohol, 1;
(4) 70% ethanol, 1;
(5) contain the aqueous solution of 0.01%DEPC, 1.
3, the detection method of a kind of synchronous detection fish infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus and viral haemorrhagic septicaemia virus is characterized in that, adopts the described test kit of claim 1, and its step is as follows,
(1) extraction of RNA template in the sample: get susceptible tissue sample homogenate or suspected virus and infect suspension 100-500 μ l, add 500-800 μ l Trizol reagent, vortex 30s, add chloroform and the primary isoamyl alcohol mixed solution of 200 μ l in ratio preparation in 24: 1, vortex 30s, the centrifugal 5-3min of 10000-15000r/min, get supernatant, it is the same centrifugal to add the equivalent aqueous isopropanol, gets precipitation, add 70% ethanolic soln of 300 μ l the same centrifugal after, remove supernatant, air-dry, add the 0.01%DEPC aqueous solution of 50 μ l, get 2-10 μ l and be used for RT-PCR;
(2) preparation of RT-PCR premix: elder generation adds each reagent in advance by following art formula and mixes, and needs the centrifugal several seconds before each reagent adds,
dNTP 1μl；
Contain Mg
2+The PCR damping fluid, 5 μ l;
Primer is to ihnf/ihnr 2 μ l;
Primer is to ipnf/ipnr, 2 μ l;
Primer is to vhsf/vhsr, 2 μ l;
Archaeal dna polymerase, 0.5 μ l;
Reversed transcriptive enzyme, 0.5 μ l;
The RNA enzyme inhibitors, 0.25 μ l;
The 0.01%DEPC aqueous solution, some;
In above-mentioned mixed solution, add respectively then:
The RNA template, 2-10 μ l gets RT-PCR premix A, cumulative volume 50 μ l;
Negative control, 5 μ l get RT-PCR premix B, cumulative volume 50 μ l;
Positive control, 5 μ l get RT-PCR premix C, cumulative volume 50 μ l;
(3) above-mentioned RT-PCR premix A, B, C are packed into PCR reaction tubes places gene-amplificative instrament, does not have the heat lid as instrument, adds the capping of 1-2 dropstone wax oil, and it is as follows that the RT-PCR loop parameter is set, and increases:
42℃×30min
95℃×4min
72℃×10min
25 ℃ * 1min or end
(4) after amplified reaction finishes, get 10-15 μ l and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 2% agarose gel electrophoresis, voltage is observed down in uv analyzer after pressing 5V/cm electrophoresis 30-40min, and negative control and positive control are correct, sample RNA extracting solution is if band occurs at 625bp, 371bp, 206bp place, then be respectively VHSV, IHNV and the IPNV positive, illustrate and carry three kinds of viruses in the testing sample, if one of them band occurs, then Dui Ying viral detected result is positive, otherwise negative.
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|---|---|---|---|
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|---|---|---|---|
| CNB2005100388874A CN100365131C (en) | 2005-04-12 | 2005-04-12 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
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| CN103233005B (en) * | 2013-05-10 | 2015-04-08 | 国家海洋局第三海洋研究所 | Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit |
| CN103525951A (en) * | 2013-10-24 | 2014-01-22 | 中国水产科学研究院黑龙江水产研究所 | LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof |
| CN105176979A (en) * | 2015-08-31 | 2015-12-23 | 孙翠言 | Method for detecting infectious pancreatic necrosis virus |
| CN105176979B (en) * | 2015-08-31 | 2018-10-26 | 施淑琴 | A method of detection infectious pancreas necrosis virus |
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| CN105441591B (en) * | 2015-12-30 | 2019-04-05 | 中国检验检疫科学研究院 | A kind of primer, kit and detection method detecting popular hematopoietic necrosis virus using pyrosequencing techniques |
| CN112176111A (en) * | 2020-11-05 | 2021-01-05 | 北京市水产技术推广站 | Primer, probe, kit and detection method for detecting infectious pancreatic necrosis virus |
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