Specific primers for detection of infectious hematopoietic necrosis virus

A technique for hematopoietic organ necrosis and specificity, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve problems such as inability to diagnose, and achieve the effects of saving time, good specificity, and sensitivity

Active Publication Date: 2014-10-01
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

Among these methods, PCR has been proved to be the fastest and most sensitive method, however, studies have shown that the nucleoprotein gene is not the most abundant gene in the IHNV genome, and using it as the target protein for virus detection is not the most sensitive method , easy to be undiagnosed when carried in the early and late stages of infection with low viral content

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  • Specific primers for detection of infectious hematopoietic necrosis virus
  • Specific primers for detection of infectious hematopoietic necrosis virus
  • Specific primers for detection 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 IHNV polymerase protein in Genbank database (accession number: L40883), use Primer Premier5.0 to select the gene conservative segment of IHNV polymerase protein to design a pair of specific primers IHNV-Lf and IHNV-Lr;

[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. After the EPC cells i...

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 IHNV polymerase protein in Genbank database (accession number: L40883), use Primer Premier5.0 to select the gene conservative segment of IHNV polymerase protein to design a pair of specific primers IHNV-Lf and IHNV-Lr;

[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 grow into dense mo...

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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 present invention relates to specific primers for infectious hematopoietic necrosis virus. Background technique [0002] Infectious haematopoietic necrosis virus (IHNV) belongs to Rhabdoviridae (Rhabdoviridae), Nora rhabdovirus (Novirhabdovirus), 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, respectively encoding viral nucleoproteins , 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 disease tha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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