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Double-PCR (polymerase chain reaction) method for detecting iridovirus of micropterus salmoides

A technology of swollen cell virus and perch rainbow, applied in the field of PCR, to achieve strong sensitivity, accurate diagnosis and species identification, and high specificity

Inactive Publication Date: 2012-12-12
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although conventional PCR and fluorescent quantitative PCR detection methods for largemouth bass virus genus have been reported, there is still a lack of a method that can quickly and accurately detect the two iridescent species of largemouth bass virus genus and swollen cell virus. virus detection method

Method used

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  • Double-PCR (polymerase chain reaction) method for detecting iridovirus of micropterus salmoides
  • Double-PCR (polymerase chain reaction) method for detecting iridovirus of micropterus salmoides
  • Double-PCR (polymerase chain reaction) method for detecting iridovirus of micropterus salmoides

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Experimental strains

[0038] Largemouth bass frog virus genus iridovirus SD-R strain, swollen cell virus genus NH-M strain, grass carp reovirus (Grass carp reovirus HZ08, GCRV HZ08), infectious hematopoietic necrosis virus (Infectious hematopoietic necrosis virus, IHNV) and yellowtail ascites virus (Yellowtail ascites virus, YTAV) were isolated, identified and preserved by the inventor; frog virus FV3 and fathead fish muscle cells (FHM) were purchased from ATCC (numbers VR-567 and CCL-42, respectively) Carp epithelial tumor cells (EPC) and grass carp kidney cells (CIK) were purchased from China Type Culture Collection (numbers GDC174 and GDC086 respectively); mandarin fish brain primary cells (SCC) were established by the inventor's laboratory according to conventional methods and save.

[0039] 2. Propagation of virus and extraction of viral DNA and RNA

[0040] Largemouth bass virus SD-R strain and FV3 were propagated by FHM, grass carp reovirus HZ08 strain was ...

Embodiment 2

[0049] 1. Collection of disease data

[0050] Fifteen largemouth bass samples were collected from Nanhai District and Shunde District of Foshan City. After the disease materials are collected, they are sent to the laboratory at low temperature.

[0051] 2. Extract DNA template

[0052] 1) Take 50 mg of spleen tissue from the above 15 fish to be tested, cut them into pieces, add 0.5 ml of lysate, digest at 55°C for 1 hour, and shake gently from time to time;

[0053] 2) Add an equal volume of phenol / chloroform / isoamyl alcohol, invert and mix well, let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, take the supernatant, extract it once with chloroform, and let stand at room temperature After 5 minutes, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant;

[0054] 3) Add 2 times the volume of absolute ethanol, let stand at room temperature for 10 minutes to precipitate DNA, and centrifuge at 12000 rpm for 10 minutes;

[0055] 4...

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Abstract

The invention discloses a double-PCR (polymerase chain reaction) method for detecting iridovirus of micropterus salmoides. By the double-PCR method, two kinds of iridoviruses belonging to ranavirus and tumefaction cell viruses can be simultaneously detected and identified by the aid of two pairs of specific primers and optimized reaction system and reaction conditions. The double-PCR method is high in sensitivity and specificity and capable of quickly and accurately diagnosing iridovirus of micropterus salmoides and identifying species.

Description

technical field [0001] The invention relates to the technical field of PCR, in particular to a double PCR technique for detecting largemouth bass iridescent virus. Background technique [0002] Largemouth bass (Micropterus salmoides, or Largemouth bass), commonly known as California perch, is native to the Mississippi River Basin in North America. An important freshwater aquaculture species in my country. Since the 1990s, the problems of largemouth bass cultured in ponds in my country have become more and more complicated, such as gill rot, enteritis, rotting skin, saprolegniasis, trichotillomaniasis, and calcitoniasis, etc., resulting in largemouth bass. The pathogens of black perch are mainly Aeromonas hydrophila, Flavobacterium columnarum, mold and parasites, and there are few reports on confirmed cases caused by virus infection. Largemouth bass iridescent virus (LMBV, also known as SCRV) generally refers to the family Iridoviridae ( Iridoviridae ) of the ranavirus genus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 王庆曾伟伟刘春李凯彬王芳王英英石存斌吴淑勤
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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