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Method for detecting shrimp white spot disease virus based on liquid chip

A technology for white spot disease and virus of prawns, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as unsuitable for a large number of sample detection, harmful to human body and environment, and cumbersome methods, etc. Achieve the effects of short time required, high sensitivity, and no environmental pollution

Inactive Publication Date: 2013-06-26
YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In situ hybridization has the advantages of strong specificity and high sensitivity, but the method is quite cumbersome and not suitable for detection of a large number of samples
The PCR method is relatively fast and sensitive, but requires ethidium bromide staining to observe the results. Ethidium bromide is a strong carcinogen, which is harmful to humans and the environment, and the problem of cross-contamination is serious

Method used

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  • Method for detecting shrimp white spot disease virus based on liquid chip
  • Method for detecting shrimp white spot disease virus based on liquid chip
  • Method for detecting shrimp white spot disease virus based on liquid chip

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the design of specific primer pair and specific probe

[0038] Through a large number of sequence alignments and comparisons of amplification effects, a specific primer pair and a specific probe for amplifying shrimp white spot disease virus were designed.

[0039]The specific primer pair is as follows (the target sequence is 232bp):

[0040] F1 (sequence 1 of the sequence listing): 5'-CCGCAATGGAAAGTCTGA-3';

[0041] R1 (SEQ ID NO: 2 of the Sequence Listing): 5'-GGTGAAGGAGGAGGTGTT-3'.

[0042] The nucleotide sequence of the specific probe (sequence 3 in the sequence listing) is as follows:

[0043] 5'-CTGATGCACAGATGAAGGAAGAAGA-3'.

Embodiment 2

[0044] Embodiment 2, application of specific primers pair of auxiliary identification prawn white spot disease virus

[0045] Shrimp white spot disease virus, epidemic hematopoietic organ necrosis virus, carp spring viremia virus, infectious hematopoietic organ necrosis virus and viral hemorrhagic sepsis virus were used as the tested viruses to carry out the following experiments:

[0046] 1. Use a DNA extraction kit to extract the genomic DNA of the virus to be tested (prawn white spot disease virus or epidemic hematopoietic organ necrosis virus).

[0047] 2. Using the genomic DNA obtained in step 1 as a template, using a primer pair composed of F1 and R1, and using a PCR kit, perform PCR amplification on a gradient PCR amplification instrument to obtain a PCR amplification product.

[0048] PCR amplification reaction system: In a 0.2mL PCR reaction tube, add 10×PCR buffer 2.5μL, dNTP (2.5mmol / L each) 2.0μL, 10pmol / μL F11μL, 10pmol / μL R11μL, Taq (5U / μL ) 0.25 μL, genomic DNA...

Embodiment 3

[0056] Embodiment 3, application primer probe composition auxiliary identification shrimp white spot disease virus

[0057] 1. Preparation of primers and probes

[0058] Prepare primers and probes as follows:

[0059] F2: 5'-biotin-CCGCAATGGAAAGTCTGA-3';

[0060] R2: 5'-biotin-GGTGAAGGAGGAGGTGTT-3'.

[0061] Probe T1: 5'-NH 2 -CTGATGCACAGATGAAGGAAGAAGA-3'.

[0062] F2 is biotinylated at the 5' end of F1, and R2 is biotinylated at the 5' end of R1. Probe T1 is obtained by amination modification at the 5' end of the single-stranded DNA fragment shown in Sequence 3 of the sequence listing.

[0063] 2. Establishment of liquid phase chip detection system

[0064] 1. Using a DNA extraction kit to extract the genomic DNA of the white spot disease virus from prawns.

[0065] 2. Using the genomic DNA obtained in step 1 as a template, using a primer pair composed of F2 and R2, and using a PCR kit, perform PCR amplification on a gradient PCR amplification instrument to obtain a PC...

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Abstract

The invention discloses a mehtod for detecting shrimp white spot disease virus based on a liquid chip. The invention provides a specific primer pair for assisting in identification of the shrimp white spot disease virus, which comprises single-stranded DNA (deoxyribonucleic acid) molecules as shown in a sequence 1 in a sequence table and the single-stranded DNA molecules as shown in a sequence 2 in the sequence table. The invention further protects a primer probe composition for assisting in identification of the shrimp white spot disease virus, which comprises the specific primer pair and a probe T1, wherein the nucleotide sequence of the probe T1 is as shown in a sequence 3 in the sequence table. The specific primer pair provided by the invention has good specificity when being used for identifying the shrimp white spot disease virus. The primer probe composition combined liquid chip provided by the invention has the advantages of good specificity, high sensitivity, simplicity and convenience in operation, short time, no environmental pollution, no threat to human health and high-throughput detection when being used for identifying the shrimp white spot disease virus. The method disclosed by the invention is very suitable for detecting import and export aquatic animals.

Description

technical field [0001] The invention relates to a pair of primers for assisting identification of white spot disease virus of prawns, a composition of primers and probes and their application, and more particularly relates to a method for detecting white spot disease virus of prawns based on a liquid phase chip. Background technique [0002] White spot disease (WSD) of prawn is one of the most serious prawn viral diseases to seawater prawn farming in the world, and it is caused by white spot syndrome virus (WSSV). White spot syndrome virus is also known as shrimp white spot disease virus. In 1993, Japan first reported the outbreak of WSD in cultured Penaeus japonicus. Subsequent studies found that WSD can not only cause disease and death of Penaeus monodon and Penaeus japonicus at various growth stages, but also cause many freshwater and seawater crustaceans, including crabs and crayfish, to become ill and die within 3-10 days of onset rate reached 100%, causing serious ec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 方绍庆尹伟力刘宁张晓文王颖许红岩段效辉粟智平耿金培李金庆
Owner YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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