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

A technology for shrimp white spot disease and virus, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as being unsuitable for the detection of a large number of samples, harmful to human body and environment, and cumbersome methods, etc. To achieve the effect of short time required, high sensitivity and no environmental pollution

Inactive Publication Date: 2015-01-14
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
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Design of specific primer pairs and specific probes

[0038] Through a large number of sequence alignments and comparison of amplification effects, specific primer pairs and specific probes for amplifying shrimp white spot virus were designed.

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

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

[0041] R1 (Sequence 2 of the Sequence Listing): 5'-GGTGAAGGAGGAGGTGTT-3'.

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

[0043] 5'-CTGATGCACAGATGAAGGAAGAAGA-3'.

Embodiment 2

[0044] Example 2. Application of specific primer pairs to assist in the identification of shrimp white spot virus

[0045] Shrimp white spot virus, epidemic hematopoietic organ necrosis virus, carp spring viremia virus, infectious hematopoietic organ necrosis virus and viral hemorrhagic septicemia virus were used as the test 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 (shrimp white spot virus or epidemic hematopoietic necrosis virus).

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

[0048] PCR amplification reaction system: In a 0.2mL PCR reaction tube, add 2.5μL of 10× PCR buffer, 2.0μL of dNTPs (2.5mmol / L each), 10pmol / μL F11μL, 10pmol / μL R11μL, Taq (5U / μL) ) 0.25 μL, genomic DNA 2.0 μL (about 300 ng), a...

Embodiment 3

[0056] Example 3. Application of primer-probe composition to assist in identifying shrimp white spot virus

[0057] 1. Preparation of primers and probes

[0058] Prepare the following primers and probes:

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

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

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

[0062] F2 was obtained by biotin labeling at the 5' end of F1, and R2 was obtained by biotin labeling 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 SEQ ID NO: 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 shrimp white spot virus.

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

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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 present invention relates to primer pairs, primer-probe compositions and their applications for assisting identification of shrimp white spot virus, and more particularly to a method for detecting shrimp white spot virus based on a liquid phase chip. Background technique [0002] White spot disease (WSD) of prawns is one of the most serious viral diseases of prawns in the world and is caused by White Spot Syndrome Virus (WSSV). White spot syndrome virus is also known as white spot disease virus of shrimp. An outbreak of WSD in farmed Penaeus japonicus was first reported in Japan in 1993. Subsequent studies have found that WSD can not only cause illness and death in P. monodon and P. japonicus at all growth stages, but also in many freshwater and marine crustaceans, including crabs and crayfish, with death within 3-10 days of onset The rate reaches 100%, causing serious economic losses. Since 1996, a large-scale outbreak of WSD in China has cause...

Claims

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Application Information

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Patent Type & Authority Patents(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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