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Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns

A detection method, the technology of WSSV-F, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems affecting the sensitivity and accuracy of test results, etc.

Active Publication Date: 2017-05-10
MARINE BIOLOGY INST OF SHANDONG PROVINCE
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Problems solved by technology

However, multiplex PCR detection is also affected by many factors, such as the activity of DNA polymerase, the quality of the template, the specificity of the designed pathogenic primers, PCR reaction conditions, and the competition of reagents between different pathogenic samples, etc. will affect the detection results. sensitivity and accuracy

Method used

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  • Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns
  • Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns
  • Multiple-PCR detection method and kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns

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Embodiment 1

[0063] Detection of shrimp white spot syndrome virus, shrimp acute hepatopancreatic necrosis (pir VP ), Shrimp hepatica, Infectious subcutaneous and hematopoietic necrosis virus test

[0064] 1) Reagent

[0065] 10×Taq Buffer: Tris-HCl 100mM, KCl 500mM, MgCl 2 20mM, dNTPs 2mM, Taq enzyme 1U / μL;

[0066] Primers: WSSPV-F, WSSV-R, AHPND-F, AHPND-R, EHP-F, EHP-R, IHHNV-F, IHHNV-R 10μM each, pure water, positive quality control DNA 10μg / ml;

[0067] The present invention is based on prawn white spot syndrome virus (White spot syndrome virus, WSSV), prawn acute hepatopancreatic necrosis disease (Acute hepatopancreatic necrosis disease, AHPND), shrimp hepatic enterocystosis (Enterocytozoon hepatopenaei, EHP), infectious subcutaneous and hematopoietic tissue necrosis The nucleic acid sequence of the virus (Infectious hypodermal and hematopoietic necrosis virus, IHHNV) was designed to detect the specificity of shrimp white spot syndrome virus, shrimp acute hepatopancreas necrosis (...

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Abstract

The invention belongs to the technical field of marine organism pathogen detection, and in particular relates to a multiple-PCR detection method and a kit for simultaneously detecting WSSV (white spot syndrome virus), AHPND (acute hepatopancreatic necrosis disease), EHP (enterocytozoon hepatopenaei) and IHHNV (infectious hypodermal and hepatopancreatic necrosis virus) in prawns. With the application of the multiple-PCR detection method, amplification of four pathogen DNAs can be simultaneously conducted in one time by virtue of special detection primers, namely WSSV F and WSSV R, AHPND F and AHPND R, EHP F and EHP R as well as IHHNV F and IHHNV R of the WSSV, the AHPND, the EHP and the IHHNV. The multiple-PCR detection method provided by the invention is convenient and rapid, low in detection limit, strong in specificity, high in sensitivity and high in accuracy rate and is capable of simultaneously detecting the white spot syndrome virus, the acute hepatopancreatic necrosis disease, the enterocytozoon hepatopenaei and the infectious hypodermal and hepatopancreatic necrosis virus in the prawns.

Description

technical field [0001] The invention belongs to the technical field of detection of marine biological pathogens, in particular to a method for simultaneously detecting white spot syndrome virus (White spot syndrome virus, WSSV), acute hepatopancreatic necrosis (AHPND) and enterocytozoon hepatopenaei. , EHP), infectious hypodermal and hematopoietic necrosis virus (Infectious hypodermal and hematopoietic necrosis virus, IHHNV) multiplex PCR detection method and kit. Background technique [0002] White spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) have been the main viruses causing outbreaks of shrimp virus diseases. The World Organization for Animal Health (OIE) classifies these two diseases as important diseases of crustaceans that must be notified to it. Acute hepatopancreatic necrosis disease (AHPND) and Enterocytozoon hepatopenaei (EHP) are the biggest threats to shrimp farming, especially Penaeus vannamei. They are two new ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/6893C12Q1/701C12Q2600/16C12Q2537/143
Inventor 许拉叶海斌盖春蕾李乐王晓璐于晓清樊英王淑娴刁菁李天保刘洪军
Owner MARINE BIOLOGY INST OF SHANDONG PROVINCE
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