CN105176979B - A method of detection infectious pancreas necrosis virus - Google Patents
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Abstract
The present invention provides a kind of method of detection infectious pancreas necrosis virus, and the sequence of used primer, upstream and downstream primer is respectively SEQ ID NO:1 and SEQ ID NO:2.The present invention devises a large amount of primers according to infectious pancreas necrosis virus genome sequence, therefrom filters out the primer that can quickly and effectively detect.Primer used in the present invention has higher specificity, sensitivity, practicability and convenience, and highly sensitive detection can be effectively carried out to infectious pancreas necrosis virus.
Description
Technical field
The invention belongs to aquatic products disease technical field of microbial detection, and in particular to a kind of detection infectious pancreatic necrosis
The method of poison.
Background technology
With the rapid development of social economy, culture fishery also shows cultivation scale and the unprecedented of cultural technique carries
Height is especially quickly grown in terms of intensive culture and industrial aquaculture, this brings nutrition and health to the life of the people.But
In the breeding process of marine fish, breaking out for viral disease often brings huge harm to aquaculture.It infects
Property Pancreatic Necrosis disease be by infectious pancreatic necrosis virus (infectious pancreatic necrosis virus,
IPNV fish infectious disease caused by), son, the juvenile fish of main harm salmon fishes, the death rate is up to 90% or more.The feature of morbidity fish
One of be seed die by visitation of God, sick fish moves about slowly first, asynergia or fair current levitating, often makees vertical revolution travelling, soon
Just it sinks under water and dead.Sick fish body color blacks, and exophthalmos, abdomen expands, and abdomen and fin ray portion are congested, and the gill is in pale red, anus
Often a long and thicker white mucus excrement is held in the palm at door.The sick fish of dissection is checked, it is seen that has ascites, sick fish pancreas to fill
Blood, pylorospasm bleeding, histocyte severe necrosis；The pale anaemia of liver,spleen,kidney, also there is a necrotic lesion, gastrorrhagia, nothing in enteron aisle
Food, and have milky or faint yellow mucus.Therefore, detect whether breeding water body, apparatus and feed are passed in time
Metachromia Pancreatic Necrosis disease virus is just extremely important.But the detection method of current IPNV be mainly histopathology technology, electron microscopy,
Cell culture technology, immunoassay technology and molecular detection technology etc..These detection methods are in accuracy, sensitivity and cost etc.
Aspect has the advantages that respective, but they are in varying degrees by needing professional and complex instrument to be limited.
Invention content
The object of the present invention is to provide a kind of methods of detection infectious pancreas necrosis virus, to make up the prior art
It is insufficient.
Present invention firstly provides a kind of primer of detection infectious pancreas necrosis virus, the sequence difference of upstream and downstream primer
For SEQ ID NO:1 and SEQ ID NO:2；
Preferably, the sequence of primer is SEQ ID NO downstream:3.
The present invention also provides a kind of kits of detection infectious pancreas necrosis virus, which includes above-mentioned draw
Object pair.
The present invention also provides a kind of methods of detection infectious pancreas necrosis virus, are to be for detecting in water body or feed
The no method for having infected infectious pancreas necrosis virus, this method include following step：
1) template of the nucleic acid of extraction detected sample as detection；
2) RNA of extraction is subjected to reverse transcription and prepares cDNA；
3) it is detected the PCR amplification of sample, 50 μ L of reaction system include Taq enzyme, the Mg of 0.05U/ μ L2+It is a concentration of
The dNTPs of 10 × PCR Buffer, 0.25mmol/L of 2mmol/L, the primer of 0.36 μm of ol/L；CDNA prepared by step 2) makees
To expand template；
The response procedures of PCR amplification are as follows：94℃ 5min；94 DEG C of 15s, 56 DEG C of 15s, 72 DEG C of 15s, 25-40 are followed
Ring；4 DEG C terminate reaction；
4) product carries out electrophoresis detection, it is determined whether has target fragment.
Positive control and negative control amplification are wherein carried out at the same time in step 3)；Positive control is that carrying infectious pancreas is bad
The plasmid of dead virus nucleic acid segment, negative control is deionized water.
The present invention devises a large amount of primers according to infectious pancreas necrosis virus genome sequence, and therefrom filtering out can be quick
The primer effectively detected.Primer used in the present invention has higher specificity, sensitivity, practicability and convenience, can have
Effect carries out infectious pancreas necrosis virus highly sensitive detection.
Specific implementation mode
The present invention is described in detail with reference to embodiment.
Embodiment 1：The design of primer
US National biology information technology center (NCBI), the infectiousness that query and search has been announced are logged in by Internet
Pancreas necrosis virus gene nucleotide series do not include unknown base content sequence, the same time same place closer to homology
Diseased plant, with Editseq the and MegAlign softwares in 7.1 software packages of DNAStar to the N gene nucleotide sequences of selected sample
It arranges into edlin, compares one by one, selected N nucleotide sequences are carried out together with Clustal W Method (software) default parameters
Source property compares, and finds the specific nucleotide sequence (target detection sequence) for characterizing the virus.
PCR amplification design of primers is carried out by Assay Design SW softwares, using the higher one group of primer of score into
Row experiment, according to the homology analysis of DNASTAR as a result, choosing sequence area more conservative in sample gene, design amplification is drawn
Object lays the foundation in order to amplify the single band of specificity for follow-up sequencing.And it is soft using design of primers to the primer of design
Part Primer_Premier_5.0 is analyzed, including△The design of primers index such as G values, primer length and G/C content selects score
Highest to be used as primer pair used in the present invention, the wherein sequence of sense primer is 5 '-CAAACAAAGCAACCGCAAC-3 '
(SEQ IDNO:1), the sequence of downstream primer is 5 '-GTCCCATTCAGGGCATAGAG-3 ' (SEQ ID NO:2), primer moves back
Fiery temperature is preferably 54 DEG C, expanding fragment length 352bp.
Embodiment 2：The specific detection of primer
In order to verify the specificity of primer of the present invention, selection carries lymphocystis disease virus LCDV, Megalocytivirus category
Irido virus (Megalocytivirus, Mega), red-spotted grouper nervous necrosis virus (red-spotted grouper
Nervous necrosis virus, RGNNV), infectious hematopoietic necrosis's poison (infectious
Haematopoietic necrosis virus, IHNV), viral hemorrhagic septicemia, VHS virus (viral hemorrhagic
Septicemia virus, VHSV) and Infectious Salmonia Anemia virus (infectious salmon anaemia virus,
ISAV) plasmid of nucleic acid fragment is as template.
The step of detection, is as follows：
(1) extraction of sample tissue nucleic acid.Sample about 0.02~1g is taken to be placed in 1.5ml plastics, with one
Sample is quickly milled to pulpous state by secondary property grinding rod, then uses DNA extraction kit (Tiangeng biochemical technology Co., Ltd, north
Capital) and TRizol (precious biotinylated biomolecule Engineering Co., Ltd, Dalian) extract DNA and RNA respectively.Concrete operation step is according to reagent
The specification of box or reagent carries out.If it is the viral sample plasmid of purchase, then the step is omitted
(2) reverse transcription prepares cDNA.When detecting the RNA virus such as RGNNV, IHNV, IPNV, VHSV, ISAV, need to RNA
Masterplate carries out reverse transcription, is carried out using the one-step method gDNA removals of Beijing Quan Shijin biotech firms and cDNA synthetic agent box.Tool
Body method is：In the microcentrifugal tube of 200 μ l, 5~500ng RNA samples, 1 μ l random primers (0.1 μ g/ μ l), 10 μ are added
L reverse transcription buffers (2 × TS Reaction Mix), 1 μ l reverse transcriptase (RT/RI Enzyme Mix), 1
μ l genomic DNAs degrading enzyme (gDNA Remover), DEPC processing water complement to 20 μ l.Above-mentioned reaction system is placed in 25 DEG C
10min, then 42 DEG C of 30min, then 85 DEG C of 5min, last 4 DEG C save backup.Detect the DNA virus such as IPNV, Mega, TRBIV
Reverse transcription is not needed, directly using the DNA masterplates of extraction.
(3) PCR amplification
In the PCR reaction tubes of single 200 μ l, the L Taq of μ containing 2.5U/ archaeal dna polymerases 0.6 μ L, 10 × PCRBuffer
(Mg2+Plus) 5 μ L, 2.5mmol/L dNTPs, 5 μ L, 2 μm of 5 μ L of ol/L Primer, the DNA profiling of extraction and the cDNA of preparation
Masterplate each 1 μ L, ddH2O is supplemented to 50 μ L.Amplified reaction program：94℃ 5min；94℃ 15s,54℃ 15s,72℃ 15s,
15 cycles；94 DEG C of 15s, 70 DEG C of 15s, 6 cycles；72℃ 3min；4 DEG C of preservations.
By product into row agarose gel electrophoresis, PCR product, 4S Red nucleic acid dye are detached using 2.0% Ago-Gel
Material is added in the gel of thawing to specifications.It takes pictures under gel imaging system after 90V constant pressure electrophoresis 35min observation.
Electrophoresis result proves that the swimming lane for there was only the sample of infectious pancreas necrosis virus has amplified production, to prove this hair
Bright primer has good specific amplification.
Embodiment 3：The measurement of primer detection sensitivity
Take 1 μ L, 10 times of gradient dilutions (104~101, 5copies/ μ L) the nucleic acid containing infectious pancreas necrosis virus matter
Grain is used as template, with the method for embodiment 2 into the measurement of line sensitivity.The result shows that primer of the invention can be expanded at 27
Cycle can detect the plasmid of 10 copies；But 5 are copied, needs to show amplified band to 38 cycles.
In order to improve sensitivity when detection, modifies optimization to downstream primer, obtained new primer sequence 5 '-
GTCCGATTCAGTGCATAGAG-3′(SEQ ID NO:3)；It is compared according to above-mentioned specificity and sensitivity detecting step
Analysis, as a result, it has been found that：Compared to the primer amplification 10ng templates before being not optimised, amplified band under the conditions of same cycle-index
It is apparent apparent.Moreover, under similarity condition, it can not be from the plasmid mould of 5 copies when the primer before optimization is recycled at 35
Amplify product that can be visual in gel electrophoresis in plate sample, and the primer after optimizing can go out in 32 cyclic amplifications it is minimum dense
The plasmid template sample that degree copies for 5, and the not appearance of primer dimer.Illustrate that the primer after optimization can significantly improve
Sensitivity, and specificity does not receive any influence.
The detection of 4 breeding water body of embodiment
The water body that infectious pancreas necrosis virus has occurred in primer pair using the present invention is detected, while to being collected into
30 tail disease fish samples carried out synchronous detection.The results show that the use of sequence being SEQ ID NO:1 sense primer and SEQ
ID NO:2 downstream primer is expanded, and purpose band is just had been detected by when all disease fish sample is recycled at 25；And water body
Being recycled at 39 can just detected.Use sequence for SEQ ID NO:3 downstream primer and SEQ ID NO:Draw 1 upstream
Object is expanded, and at 35, cycle can obtain amplification and obtain positive band；So as to avoid the generation of false negative result.
Claims (2)
1. a kind of primer pair of detection infectious pancreas necrosis virus, which is characterized in that the upstream and downstream primer of the primer pair
Sequence be respectively SEQ ID NO:1 and SEQ ID NO:3.
2. a kind of kit of detection infectious pancreas necrosis virus, which is characterized in that the kit includes to have the right to want
Seek the primer pair described in 1.
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| CN201510548476.3A CN105176979B (en) | 2015-08-31 | 2015-08-31 | A method of detection infectious pancreas necrosis virus |
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| CN201510548476.3A CN105176979B (en) | 2015-08-31 | 2015-08-31 | A method of detection infectious pancreas necrosis virus |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687447A (en) * | 2005-04-12 | 2005-10-26 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101906486A (en) * | 2010-08-03 | 2010-12-08 | 中国水产科学研究院黄海水产研究所 | Gene chip for detecting various fish pathogens and detecting method thereof |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
-
2015
- 2015-08-31 CN CN201510548476.3A patent/CN105176979B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687447A (en) * | 2005-04-12 | 2005-10-26 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101906486A (en) * | 2010-08-03 | 2010-12-08 | 中国水产科学研究院黄海水产研究所 | Gene chip for detecting various fish pathogens and detecting method thereof |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
Non-Patent Citations (3)
| Title |
|---|
| AF160258.1;Genbank;《Genbank》;20030618;序列 * |
| 传染性胰脏坏死病毒逆转录聚合酶链式反应法（RT-PCR）的研究;吴斌 等;《中国动物检疫》;20121231;第29卷(第9期);摘要，表1，1材料与方法部分，2结果与讨论部分特别是2.2-2.3 * |
| 实时荧光定量RT-PCR检测鱼类传染性胰脏坏死病病毒方法的建立;徐晔 等;《安徽农业科学》;20111231;第39卷(第31期);摘要，2结果与分析部分 * |
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