Method for detecting spring viremia of carp virus based on liquid chip

A viremia and virus technology, applied in the field of detection of carp spring viremia virus based on liquid phase chips, can solve the problems of cross-contamination, cumbersome operation, long detection cycle, etc., achieve good specificity, do not pollute the environment, and require time short effect

Inactive Publication Date: 2013-06-26
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytological diagnostic techniques mainly include the use of cell culture to isolate viruses, histopathological sections, and electron microscope observation, which are cumbersome to operate, have a long detection cycle, and have low sensitivity
Immunological diagnostic techniques mainly include immunofluorescence detection and immune dot hybridization, which have the advantages of strong specificity and high sensitivity, but the steps are quite cumbersome and are not suitable for detection of a large number of samples
Molecular biology diagnostic techniques mainly include polymerase chain reaction (PCR), which is fast and sensitive, but requires agarose gel electrophoresis and staining with ethidium bromide to observe the results. Ethidium bromide is a carcinogen and is harmful to the human body and the environment. Hazardous, cross-contamination problem is more serious

Method used

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  • Method for detecting spring viremia of carp virus based on liquid chip
  • Method for detecting spring viremia of carp virus based on liquid chip
  • Method for detecting spring viremia of carp virus based on liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0038] A specific primer pair and a specific probe for amplifying the coding gene of the glycoprotein of carp spring viremia virus are designed through a large number of sequence alignments and comparisons of amplification effects.

[0039] The specific primer pair is as follows (the target sequence is 153bp):

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

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

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

[0043] 5'-TGTATATAAAGGAAAGGATGGGAAG-3'.

Embodiment 2

[0044] Example 2, application of specific primer pairs to aid in the identification of carp spring viremia virus

[0045] Carp spring viremia virus, infectious hematopoietic necrosis virus, grass carp hemorrhagic disease virus, viral hemorrhagic sepsis virus and infectious pancreatic necrosis virus were used as the tested viruses to carry out the following experiments:

[0046] 1. Use the RNA extraction kit to extract the total RNA of the virus to be tested.

[0047] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F1 and R1, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PCR amplification products.

[0048] RT-PCR amplification reaction system: In a 0.2mL PCR reaction tube, add 10×RT-PCR buffer 2.5μL, dNTP (each 2.5mmol / L) 2.0μL, 10pmol / μL F1 1μL, 10pmol / μL R1 1μL , Inhibiter 0.5μL, AMV XL 0.5μL, AMV Taq (5U / μL) 0.25μL, 25mmol / L MgCl 2 5.0 μL, 5.0 μL total RNA (about 300 ...

Embodiment 3

[0052] Example 3, application of primer probe composition to assist in the identification of carp spring viremia virus

[0053] 1. Preparation of primers and probes

[0054] Prepare primers and probes as follows:

[0055] F2: 5'-biotin-TTGGGATGACTGGGAGTT-3';

[0056] R2: 5'-biotin-GGGATAATATCGGCTTGG-3'.

[0057] Probe T1: 5'-NH 2 -TGTATATAAAGGAAAGGATGGGAAG-3'.

[0058] 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.

[0059] 2. Establishment of liquid phase chip detection system

[0060] 1. Use the RNA extraction kit to extract the total RNA of carp spring viremia virus.

[0061] 2. Using the total RNA obtained in step 1 as a template, using a primer pair composed of F2 and R2, and using an RT-PCR kit, perform RT-PCR amplification on a gradient PCR amplification instrument to obtain RT-PCR ampli...

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PUM

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Abstract

The invention discloses a mehtod for detecting spring viremia of carp virus based on a liquid chip. The invention provides a specific primer pair for assisting in identification of the spring viremia of the carp 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 spring viremia of the carp 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 spring viremia of the carp 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 spring viremia of the carp 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 primer pair, a primer-probe composition and their application for assisting in the identification of carp spring viremia virus, and more particularly relates to a method for detecting carp spring viremia virus based on a liquid phase chip. Background technique [0002] Spring viraemia of carp (SVC), also known as spring viraemia of carp, is an acute, hemorrhagic infectious disease that must be declared to the World Organization for Animal Health (OIE). Carp spring viremia is caused by Spring viraemia of carp virus (SVCV) virus. Carp spring viremia virus is also called carp rhabdovirus (Rhabdovirus carpio). [0003] SVCV can infect a variety of fish such as koi, silver carp, bighead carp, crucian carp, grass carp and pike, but is most sensitive to carp, which is the main host of SVCV. Carp of any age in fish farms can suffer from SVC. The high and low water temperature during the alternation of winter and spring reduces the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 方绍庆贾鹏孙明君史秀杰尹伟力刘宁
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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