Method for preparing positive control for single-tube RT-PCR detection of three fish disease virus

A technology for viral hemorrhage and viruses, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as high requirements, complicated and cumbersome operation steps, and long time

Inactive Publication Date: 2006-05-24
LIANYUN PORT IMMIGRATION INSPECTION & QUARANTINE BUREAU PEOPLES REPUBLIC OF CHINA
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  • Claims
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Problems solved by technology

At present, the commonly used detection method for the three fish diseases is to inoculate sensitive fish and cells with disease materials for virus propagation, isolate and purify the virus, and then use serum neutralization, immunofluorescence and enzyme-linked immunoassay methods for virus identification. The operation steps are complicated and tedious , up to 2-4 weeks
Since these three viruses are all RNA viruses, using virus-infected materials to extract RNA fo...

Method used

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  • Method for preparing positive control for single-tube RT-PCR detection of three fish disease virus
  • Method for preparing positive control for single-tube RT-PCR detection of three fish disease virus

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Embodiment 1

[0081] Example 1. Refer to attached picture. A method for preparing positive controls for single-tube RT-PCR detection of viral hemorrhagic sepsis virus, fish infectious hematopoietic organ necrosis virus and infectious pancreatic necrosis virus, respectively selecting the G gene of viral hemorrhagic sepsis virus , the VP gene of fish infectious hematopoietic necrosis virus and the N gene of infectious pancreatic necrosis virus were used as the target gene fragments, and the target gene fragments were inserted into the pUC57 plasmid vector, and then transfected into DH5α Escherichia coli for large-scale expression in vitro, respectively. Fragments of 625bp, 371bp, and 206bp were amplified, among which,

[0082] The sequence of the inserted VHSV target gene fragment is:

[0083] agggaagatt cctttgtccc gattcgacca gctcaactca ggtgtcctca

[0084] tgaatttgaa gacataaaca agggactggt ttccgtccca actcagatca

[0085] tccatctccc gctatcagtc accagcgtct ccgcagtagc gagtggccac

[0086] tacct...

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Abstract

The invention relates to a method of make single valve RT-PCR dectecting masculine comparing virulence haemorrhagic septicemia virus, fish infectivity hemocytopoietic organ putrescence and infectivity pancreas putrescence germina. Its feature is selecting G gene from virulence haemorrhagic septicemia virus, VP gene from fish infectivity hemocytopoietic organ putrescence and N gene from infectivity pancreas putrescence germina, inserting into pUC57 particle carrier and transfect to DH5 alpha coliform bacteria to take in vitro expression, and extending the section 625bp, 371bp, 206bp. It has very high use value.

Description

technical field [0001] The invention relates to a method for preparing a positive control for detecting aquatic animals, especially fish viruses, in particular to a single tube of viral hemorrhagic sepsis virus, fish infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus Preparation method of positive control for RT-PCR detection. Background technique [0002] Viral hemorrhagic septicemia virus (VHSV), fish infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are listed as the first and second class infectious diseases of imported animals in the People's Republic of China. VHS and IHN are the second-class and third-class infectious diseases stipulated in the "Animal Epidemic Prevention Law of the People's Republic of China" respectively. These three diseases are listed in the OIE list of the International Office of Epizootics. Both IHN and VHS viruses belong to the single-stranded RNA vi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/70C12P19/34C12Q1/04
Inventor 段宏安张睿姚燕林王海涛周毅李金华刘小琴
Owner LIANYUN PORT IMMIGRATION INSPECTION & QUARANTINE BUREAU PEOPLES REPUBLIC OF CHINA
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