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Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit

A hematopoietic organ necrosis and RT-PCR technology, applied in the field of RT-PCR detection kits, can solve the problems of cumbersome process and no kits, and achieve the effect of simple operation and complete reagents

Active Publication Date: 2013-08-07
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There were also reports about IHNVRT-PCR detection methods before, but only specific primers were reported, and no kit was formed. It was necessary to purchase a large number of supporting reagents during operation, and the process was cumbersome.

Method used

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  • Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit
  • Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0054] The preparation method of the positive control is as follows:

[0055] Step 1, primer design, the specific method is:

[0056] According to the obtained IHNV nucleoprotein gene sequence, design the following two primers, ihn-long-F and ihn-long-R, for constructing recombinant plasmid pSP-IHNV for in vitro transcription of IHNV nucleoprotein gene RNA fragment, carrying HindⅢ and BamHI enzymes respectively Cutting point:

[0057] ihn-long-F: its nucleotide sequence is shown in SEQ ID NO.3;

[0058] ihn-long-R: its nucleotide sequence is shown in SEQ ID NO.4.

[0059] Step 2, the construction of the recombinant plasmid pSP-IHNV containing the detection target fragment and the preparation of the RNA fragment as a positive control, the specific steps are:

[0060] 1) Construction of pSP-IHNV recombinant plasmid

[0061] Using ihn-long-R as a reverse transcription primer, the virus RNA sample was used as a template, and the target fragment was obtained through reverse tra...

Embodiment 1

[0099] Example 1 Specificity experiment of primers

[0100] In order to determine the specificity of the RT-PCR detection kit, use other viral RNA or DNA as templates, and perform detection according to the method of use of the kit. The other viruses include pancreatic necrosis virus (Infectious pancreatic necrosis virus, IPNV), Viral haemorrhagic septicemia virus (VHSV) and carp pure blood necrosis virus (Spring viremia of carp virus, SVCV). The experimental results showed that except for infectious hematopoietic necrosis virus, the target fragment of 208bp could not be detected after agarose gel electrophoresis for other viruses (see figure 2 ). It shows that the primers used in the detection kit have good specificity.

Embodiment 2

[0101] Embodiment 2 Sensitivity detection experiment

[0102] Take 2 μL of different concentration gradients (1×10 7 —1×10 2 copies / μL) of the positive control RNA fragment was used as a template to detect according to the method used in the kit, and then use 1.5% agarose gel electrophoresis to detect the results, the results are shown (see image 3 ), the method can detect about 10 2 positive RNA, equivalent to 10 2 virus particles.

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Abstract

The invention provides a reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and a preparation method of the kit, and relates to an RT-PCR detection kit. The RT-PCR detection kit for the IHNV is provided with a kit body, an operating instruction and detection reagents, wherein the operating instruction and the detection reagents are arranged in the kit body; and the detection reagents comprise lysate, adsorption liquid, a washing solution, diethyl pyrocarbonate (DEPC) treating water, reverse transcription reaction liquid, IHNV-PCR reaction liquid, position control and negative control. The preparation method comprises the following steps of: preparing the kit body; preparing the detection reagents; and placing the operating instruction, the lysate, the adsorption liquid, the washing solution, the DEPC treating water, the reverse transcription reaction liquid, the IHNV-PCR reaction liquid, the position control and the negative control into the kit body to obtain the RT-PCR detection kit for the IHNV. The reagents are complete and the operation is simple.

Description

technical field [0001] The invention relates to a RT-PCR detection kit, in particular to an RT-PCR detection kit for infectious hematopoietic necrosis virus (IHNV) and a preparation method thereof. Background technique [0002] Infectious hematopoietic necrosis virus (IHNV) is a serious infectious disease of salmon and trout. IHNV belongs to the genus Novihabdovirus of the family Rhabdoviridae. IHNV is the representative species of this genus, and WRAC (ATCC code is VR-1392) is the representative strain of IHNV. IHNV was only prevalent in North America and some European countries in the early days. In 1968, it was introduced to Hokkaido, Japan from Alaska along with sockeye salmon eggs. With the increase of import and export trade of aquatic animals and their products, IHNV has been introduced into our country and is prevalent in some areas, causing heavy losses to the aquatic industry. [0003] Reverse transcription chain polymerase reaction (RT-PCR) is the most commonly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68C12R1/93
Inventor 陈新华敖敬群胡国海母尹楠
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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