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High-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus

A technology for hematopoietic organ necrosis and detection methods, which is applied in the direction of microbial measurement/testing, biochemical equipment and methods, etc., can solve the problem of low sensitivity and achieve low sensitivity, sensitivity, and good specificity

Active Publication Date: 2013-06-19
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The present invention aims to solve the problem of low sensitivity of the existing PCR method for detecting infectious hematopoietic organ necrosis virus, and provide an efficient PCR detection method for infectious hematopoietic organ necrosis virus

Method used

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  • High-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus
  • High-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus
  • High-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus

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specific Embodiment approach 1

[0021] Specific embodiment one: the high-efficiency PCR detection method of infectious hematopoietic necrosis virus of the present embodiment is carried out according to the following steps:

[0022] 1. According to the gene sequence of the IHNV polymerase protein in the Genbank database (accession number: L40883), a pair of specific primers IHNV-Lf and IHNV-Lr were designed using Primer Premier 5.0 to select the gene conservative segment of the IHNV polymerase protein;

[0023] 2. Take the disease material to be tested from fish without clinical symptoms of infectious hematopoietic organ necrosis, put it in a small homogenizer, grind on ice for 3-5 minutes, then centrifuge at 3000g for 5 minutes, discard the precipitate, and the supernatant is passed through 0.22 Filter and sterilize with a μM disposable filter to obtain a sterile homogenate supernatant, and then use MEM medium to dilute the sterile homogenate supernatant 1000 times to obtain a tissue filtrate;

[0024] 3. Af...

specific Embodiment approach 2

[0035] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the reaction system of PCR amplification in step four is a reaction system of 25 μL, which consists of the following components:

[0036]

[0037]

[0038] PCR amplification conditions were: 95°C pre-denaturation for 5 min, 95°C denaturation for 1 min, 50°C annealing for 50 s, 72°C extension for 1 min, a total of 30 cycles, 72°C extension for 10 min, and 4°C to end the reaction. Other steps and parameters are the same as those in Embodiment 1.

Embodiment

[0040] The efficient PCR detection method of infectious hematopoietic necrosis virus is carried out according to the following steps:

[0041] 1. According to the gene sequence of the IHNV polymerase protein in the Genbank database (accession number: L40883), a pair of specific primers IHNV-Lf and IHNV-Lr were designed using Primer Premier 5.0 to select the gene conservative segment of the IHNV polymerase protein;

[0042] 2. Take the disease material to be tested from fish without clinical symptoms of infectious hematopoietic organ necrosis, put it in a small homogenizer, grind on ice for 3-5 minutes, then centrifuge at 3000g for 5 minutes, discard the precipitate, and the supernatant is passed through 0.22 Filter and sterilize with a μM disposable filter to obtain a sterile homogenate supernatant, and then use MEM medium to dilute the sterile homogenate supernatant 1000 times to obtain a tissue filtrate;

[0043] 3. After the EPC cells in the culture flask are subcultured to...

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Abstract

The invention discloses a high-efficient PCR (polymerase chain reaction) detection method of infectious hematopoietic necrosis virus and relates to a detection method of the infectious hematopoietic necrosis virus. The invention aims at the problem that the existing PCR method for inspecting the infectious hematopoietic necrosis virus is not high in sensitivity. The method comprises the following steps: 1) designing specific primers IHNV-Lf and IHNV-Lr; 2) getting a tissue filtrate after treatment of a detected pathological material; 3) preparing RNA (ribonucleic acid) of the IHNV (infectious hematopoietic necrosis virus); 4) performing PCR amplification to get an amplified product; and 5) observing the PCR amplified product and determining a result to end the detection. The detection method disclosed by the invention has great specificity, and the cross reaction with VHSV (viral hemorrhagic septicemia virus) does not exist; and according to the detection method disclosed by the invention, a polymerase protein is taken as a detection target, the specific primers IHNV-Lf and IHNV-Lr are utilized for detection, the operation is simple, the accuracy is high, the detection method is sensitive, the sensitivity under the condition of low concentration is great, the detection cost is relatively lower, and the detection method is more time-saving in comparison with an existing nested PCR (two-round PCR).

Description

technical field [0001] The invention relates to a detection method for infectious hematopoietic organ necrosis virus. Background technique [0002] Infectious haematopoietic necrosis virus (IHNV) belongs to Rhabdoviridae (Rhabdoviridae), Nora Rhabdovirus (Novirhabdovirus), and IHNV is the representative species of this genus. IHNV virus particles are bullet-shaped, enveloped, and unstable to heat, acid, and ether. The IHNV virion contains a linear, antisense, single-stranded RNA, and its genome structure contains six genes, N-P(M1)-M2-G-NV-L, from the 3' end to the 5' end, which encode viral nucleoproteins respectively , phosphoproteins, matrix proteins, glycoproteins, nonstructural proteins and polymerase proteins, the current genome sequencing shows that the full length of the IHNV genome is about 11kb. [0003] Infectious hematopoietic necrosis (IHN) is an acute systemic infectious disease of fish. The World Organization for Animal Health (OIE) lists it as an animal dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 徐黎明卢彤岩刘红柏
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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