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RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof

A technology of RT-PCR and viral hemorrhage, which is applied in the field of RT-PCR detection kit and its preparation of viral hemorrhagic sepsis virus, can solve the problems of cumbersome process and no kit, and achieve simple operation and complete reagents Effect

Active Publication Date: 2014-11-26
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There were also reports about the VHSVRT-PCR detection method 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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  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof
  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof
  • RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] The specificity experiment of embodiment 1 primer

[0083] In order to determine the specificity of the RT-PCR detection kit, use other viral RNA or DNA as a template respectively, and detect according to the method of use of the kit, and the other viruses include infectious hematopoietic necrosis virus (Infectious hematopoietic necrosis virus, IHNV), Infectious pancreatic necrosis virus (IPNV) and Spring viremia of carp virus (SVCV). The experimental results showed that except for the viral hemorrhagic sepsis virus, the target fragment of 471bp 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

[0084] Embodiment 2 Sensitivity detection experiment

[0085] Take 2 μL of different concentration gradients (1×10 6 —1×10 1 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.

Embodiment 3

[0086] Embodiment 3 recovery rate experiment

[0087] To analyze the efficiency of viral RNA extraction from the samples, recovery experiments were performed. 20 μL of 1 x 10 6 , 1×10 5 , 1×10 4 The copy / μL positive control RNA was mixed with the negative sample, and the RNA was extracted according to the usage method of the kit, and the RNA was dissolved in 20 μL DEPC-treated water. Then use 2 μL of each concentration gradient positive control RNA and newly extracted RNA as templates for detection. The agarose gel electrophoresis detection result shows that the recovery rate of this extraction method is greater than 50% (see Figure 4 ).

[0088]

[0089]

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Abstract

The invention discloses a RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and a preparation method thereof, and relates to an RT-PCR detection kit. The RT-PCR detection kit for viral haemorrhagic septicaemia virus is provided with a kit body, an operation instruction and a detection reagent, wherein the operation instruction and the detection reagent are arranged in the kit body; and the detection reagent comprises a lysis solution, an adsorption solution, a washing solution, DEPC treatment water, an inverse transcription reaction solution, a VHSV-PCR reaction solution, a positive control and a negative control. The preparation method comprises the following steps of: preparing the detection reagent; and putting the operation instruction, the lysis solution, the adsorption solution, the washing solution, the DEPC treatment water, the inverse transcription reaction solution, the VHSV-PCR reaction solution, the positive control and the negative control into the kit body to obtain the RT-PCR detection kit for viral haemorrhagic septicaemia virus. The reagent is complete, and the operation is simple.

Description

technical field [0001] The invention relates to a RT-PCR detection kit, in particular to a RT-PCR detection kit for viral hemorrhagic septicemia virus (VHSV) and a preparation method thereof. technical background [0002] Viral hemorrhagic septicemia (VHS) is a fatal infectious disease infecting salmon, trout, turbot, perch, desert flounder, brown trout, grayling and other fish. The disease causes haemorrhages in the fish's skin, kidneys and liver, and the mortality rate is about 90%. The causative agent of VHS is viral hemorrhagic sepsis virus (VHSV), which belongs to the Novirhabdovirus genus of the Rhabdoviridae family. The VHSV genome is a single-stranded negative-strand RNA with a length of about 12kp. The linear genome encodes 5 proteins, including: nucleocapsid protein (N protein), two structural proteins (P and M proteins), and glycoprotein (G protein). , RNA polymerase (L protein) and a nonstructural protein (NV protein), the sequence is 3'-Leader-N-P-M-G-NV-L-Tra...

Claims

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Application Information

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