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

A hematopoietic organ necrosis and infectivity technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Polluted environment, short time required, good specific effect

Inactive Publication Date: 2013-06-26
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • 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 infectious haematopoietic necrosis virus based on liquid-phase chip
  • Method for detecting infectious haematopoietic necrosis virus based on liquid-phase chip
  • Method for detecting infectious haematopoietic necrosis virus based on liquid-phase 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 infectious hematopoietic organ necrosis virus are designed through a large number of sequence alignments and amplification effect comparisons.

[0039] The specific primer pair is as follows (the target sequence is 126bp):

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

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

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

[0043] 5'-TCCTCCGACTTGACTCACCGC-3'.

Embodiment 2

[0044] Example 2, application of specific primer pairs to aid in the identification of infectious hematopoietic organ necrosis virus

[0045] Infectious hematopoietic necrosis virus, viral hemorrhagic sepsis virus, carp spring viremia virus, infectious pancreatic necrosis virus and viral neuronecrosis virus were used as the viruses to be tested for 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 (2.5mmol / L each) 2.5μL, 10pmol / μL F10.5μL, 10pmol / μL R10 .5μL, Inhibiter0.5μL, AMV XL0.5μL, AMV Taq (5U / μL)0.5μL, 25mmol / L MgCl 2 5.0 μL, total RNA 3.0 μL (about 3...

Embodiment 3

[0052] Example 3, application of primer probe composition to assist in the identification of infectious hematopoietic necrosis virus

[0053] 1. Preparation of primers and probes

[0054] Prepare primers and probes as follows:

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

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

[0057] Probe T1: 5'-NH 2 -TCCTCCGACTTGACTCACCGC-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 infectious hematopoietic organ necrosis 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 in...

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Abstract

The invention discloses a method for detecting an infectious haematopoietic necrosis virus based on liquid-phase chip. The invention provides a specific primer pair for identifying the infectious 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 infectious 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 infectious haematopoietic necrosis virus. The primer probe composition provided by the invention is combined with the liquid-phase chip to identify the infectious 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 invention relates to a primer pair, a primer probe composition and their application for assisting in the identification of infectious hematopoietic organ necrosis virus, in particular to a method for detecting infectious hematopoietic organ necrosis virus based on a liquid phase chip. Background technique [0002] Infectious hematopoietic necrosis (IHN) is a second-class infectious disease listed in the "List of Class I and Class II Infectious Diseases and Parasitic Diseases of Imported Animals of the People's Republic of China" It is an important epidemic disease in the OIE list of the International Office of Epizootics. [0003] Fish infectious hematopoietic necrosis is caused by infectious hematopoietic necrosis virus (IHNV). IHNV is an RNA virus, a member of the Rhabdoviridae family, approximately 120-180 nm long. IHNV can infect a variety of seawater and freshwater cultured fish, and is prevalent in Europe, the United States, Japan, Korea a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 尹伟力郑小龙方绍庆贾鹏刘宁王颖
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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