Method for detecting infectious pancreatic necrosis virus

A pancreas necrosis virus, infectious technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve highly sensitive detection and high specificity.

Active Publication Date: 2015-12-23
浙江恒驭生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These detection methods have their own advantages in terms of accuracy, sensitivity, and cost, but they are limited by the need for professionals and complex instruments to varying degrees.

Method used

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  • Method for detecting infectious pancreatic necrosis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the design of primer

[0017] Log in to the National Center for Bioinformatics (NCBI) of the United States through the Internet to query and retrieve the published nucleotide sequence of the infectious pancreatic necrosis virus gene, which does not contain unknown base component sequences. For diseased plants, use the Editseq and MegAlign software in the DNAStar7.1 software package to edit the N gene nucleotide sequence of the selected sample, compare them one by one, and use the default parameters of ClustalWMethod (software) to carry out the selected N nucleotide sequence Homology comparison to find the specific nucleotide sequence (target detection sequence) that characterizes the virus.

[0018] PCR amplification primers are designed by AssayDesignSW software, and a set of primers with higher scores are used for experiments. According to the homology analysis results of DNASTAR, the more conservative sequence regions in the sample genes are selected, a...

Embodiment 2

[0019] Embodiment 2: the specific detection of primer

[0020] In order to verify the specificity of the primers of the present invention, select those carrying lymphocyst virus LCDV, cytomegalovirus iridovirus (Megalocytivirus, Mega), red-spotted groupernervous necrosis virus (red-spottedgroupernervousnecrosisvirus, RGNNV), infectious hematopoietic organ Necrosis virus (infectious shaematopoietic necrosis virus, IHNV), viral hemorrhagic septicemia virus (viral hemorrhagic septicemia virus, VHSV) and infectious salmon anemia virus (infectious salmon anemia virus, ISAV) nucleic acid fragment plasmid as a template.

[0021] The detection steps are as follows:

[0022] (1) Extraction of sample tissue nucleic acid. About 0.02-1 g of the sample was placed in a 1.5 ml plastic microcentrifuge tube, and the sample was quickly ground into a slurry with a disposable grinding rod, and then DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing) and TRizol (Baobao) were us...

Embodiment 3

[0028] Embodiment 3: the mensuration of primer detection sensitivity

[0029] Take 1 μL of 10-fold serial dilution (10 4 ~10 1 , 5copies / μL) plasmid containing infectious pancreatic necrosis virus nucleic acid as a template, the method of Example 2 was used to measure the sensitivity. The results show that the primers of the present invention can detect 10 copies of the plasmid in 27 amplification cycles; but for 5 copies, it takes 38 cycles to display the amplified band.

[0030] In order to improve the sensitivity of detection, the downstream primers were modified and optimized to obtain a new primer sequence 5'-GTCCGATTCAGTGCATAGAG-3' (SEQ ID NO: 3); comparative analysis was carried out according to the above-mentioned specificity and sensitivity detection steps, and it was found that: Compared with unoptimized primers to amplify 10ng template, the amplified bands are clearer and more obvious under the same cycle number conditions. Moreover, under the same conditions, pr...

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Abstract

The invention provides a method for detecting an infectious pancreatic necrosis virus. The sequences of upstream and downstream primers adopted by the method are respectively shown as SEQ ID NO:1 and SEQ ID NO:2. According to the invention, a large number of primers are designed according to the genomic sequences of the infectious pancreatic necrosis virus, and the primers capable of rapidly and effectively detecting are screened. The primers used by the method disclosed by the invention have the characteristics of high specificity, high sensitivity, high practicability and high convenience, and can carry out highly-sensitive detection on the infectious pancreatic necrosis virus.

Description

technical field [0001] The invention belongs to the technical field of detecting aquatic disease microorganisms, and in particular relates to a method for detecting infectious pancreatic necrosis virus. Background technique [0002] With the rapid development of the social economy, the aquaculture industry has also shown an unprecedented improvement in the scale of farming and farming technology, especially in the intensive farming and factory farming, which has brought nutrition and health to people's lives. However, during the aquaculture process of marine economic fish, the outbreak of viral diseases often brings great harm to the aquaculture industry. Infectious pancreatic necrosis virus (IPNV) is a fish infectious disease caused by infectious pancreatic necrosis virus (IPNV), which mainly harms the larvae and juveniles of salmonids, with a mortality rate of more than 90%. One of the characteristics of the diseased fish is the sudden death of the seedlings. The diseased...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
Inventor 孙翠言张培类延乐
Owner 浙江恒驭生物科技有限公司
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