Method for detecting epizootic haematopoietic necrosis virus based on liquid-phase chip

A hematopoietic organ necrosis and epidemic technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. The effect of stability, short time required, and no environmental pollution

Inactive Publication Date: 2014-12-17
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

The operation of immunofluorescence is cumbersome, the detection cycle is long, and the sensitivity is low
ELISA needs to use imported detection reagents, imported reagents and kits are very expensive, and must be ordered about 2 months in advance when used, which will seriously affect the detection process
Nucleic acid hybridization has the advantages of strong specificity and high sensitivity, but it is quite cumbersome and not suitable for detection of a large number of samples
PCR is relatively fast and sensitive, but it needs agarose gel electrophoresis and ethidium bromide staining to observe the results. Ethidium bromide is a carcinogen, which is harmful to human body and environment, and the problem of cross contamination is serious

Method used

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  • Method for detecting epizootic haematopoietic necrosis virus based on liquid-phase chip
  • Method for detecting epizootic haematopoietic necrosis virus based on liquid-phase chip
  • Method for detecting epizootic haematopoietic necrosis virus based on liquid-phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Design of specific primer pairs and specific probes

[0037] Through a large number of sequence alignments and comparisons of amplification effects, specific primer pairs and specific probes for the amplification of epidemic hematopoietic necrosis virus are designed.

[0038] The specific primer pairs are as follows (the target sequence is 179bp):

[0039] F1 (Sequence 1 of the Sequence Listing): 5’-AACACGGCATACCTGGAC-3’;

[0040] R1 (sequence 2 of the sequence listing): 5'-GAGAAGAAGAATGGGAGGG-3'.

[0041] The nucleotide sequence of the specific probe (sequence 3 in the sequence list) is as follows:

[0042] 5'-AGTACACCATGCCAGAGGCCAAGCG-3'.

Embodiment 2

[0043] Example 2: Application of specific primer pairs to assist in the identification of epidemic hematopoietic organ necrosis virus

[0044] The epidemic hematopoietic organ necrosis virus, prawn white spot virus, carp spring viremia virus, infectious hematopoietic organ necrosis virus and viral hemorrhagic sepsis virus were used as the virus to be tested for the following experiments:

[0045] 1. Use the DNA extraction kit to extract the genomic DNA of the virus to be tested (epidemic hematopoietic necrosis virus or prawn white spot virus).

[0046] 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, perform PCR amplification on a gradient PCR amplification machine to obtain PCR amplification products.

[0047] PCR amplification reaction system: in 0.2mL PCR reaction tube, add 10×PCR buffer 2.5μL, dNTP (2.5mmol / L each) 2.0μL, 10pmol / μL F1 1μL, 10pmol / μL R1 1μL, Taq(5U / μL) 0.25μL, genomic DNA 2.0μL (about 300ng), ad...

Embodiment 3

[0055] Example 3. Application of primer probe composition to assist identification of epidemic hematopoietic organ necrosis virus

[0056] 1. Preparation of primers and probes

[0057] Prepare the following primers and probes:

[0058] F2: 5’-biotin-AACACGGCATACCTGGAC-3’;

[0059] R2: 5’-biotin-GAGAAGAAGAATGGGA GGG-3’.

[0060] Probe T1: 5'-NH2-AGTACACCATGCCAGAGGCCAAGCG-3'.

[0061] F2 is biotin-labeled at the 5'end of F1, and R2 is biotin-labeled 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.

[0062] 2. Establishment of liquid-phase chip detection system

[0063] 1. Use DNA extraction kit to extract genomic DNA of epidemic hematopoietic organ necrosis virus.

[0064] 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, perform PCR amplification on a gradient PCR amplification machine to obtain PCR amplification...

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Abstract

The invention discloses a method for detecting an epizootic haematopoietic necrosis virus based on liquid-phase chip. The invention provides a specific primer pair for identifying the epizootic haematopoietic necrosis virus in an auxiliary manner, wherein the specific primer pair is composed of a single-chain DNA (Deoxyribonucleic Acid) molecule as shown in sequence 1 of a sequence table and the single-chain DNA molecule as shown in sequence 2 of the sequence table. The invention further protects a primer probe composition for identifying the epizootic haematopoietic necrosis virus in the auxiliary manner, wherein the primer probe composition is composed of the specific primer pair and a probe T1; and a nucleotide sequence of the probe T1 is as shown in sequence 3 of the sequence table. The specific primer pair provided by the invention has good specificity in identifying the epizootic haematopoietic necrosis virus. The primer probe composition provided by the invention is combined with the liquid-phase chip to identify the epizootic haematopoietic necrosis virus, and therefore, the method has the advantages of being good in specificity, high in sensitivity, simple to operate, short in needed time, free of environmental pollution, free of health risks to the people and capable of carrying out high-flux detection.

Description

Technical field [0001] The present invention relates to a primer pair, a primer probe composition and their applications for assisting in identifying epidemic hematopoietic organ necrosis virus, and in particular to a method for detecting epidemic hematopoietic organ necrosis virus based on a liquid phase chip. Background technique [0002] Epizootic hematopoietic necrosis (EHN) is an infectious fish disease that causes visceral necrosis in juvenile and adult redfin perch, catfish, rainbow trout, and catfish, and even death. Epidemic hematopoietic organ necrosis is caused by epidemic hematopoietic necrosis virus (EHNV). EHN usually occurs in spring or early summer. The symptoms are as follows: fish swim abnormally, sometimes spiraling to the surface of the water body; more bleeding points appear at the base of the fin rays, the head is red, and the muscles near the bones are white; the liver is more frequent A gray-white lesion with a diameter of 1-3mm; the spleen is red, swolle...

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 方绍庆尹伟力王颖赵玉然刘宁许红岩段效辉耿金培李金庆
Owner YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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