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Subtype H5 avian influenza virus detection kit and use thereof

A bird flu virus and kit technology, applied in the field of H5 subtype bird flu virus detection kits, can solve the problems of complicated process, long time-consuming, expensive fluorescent probes, etc., and achieve high specificity, high sensitivity, simple and convenient operation Effect

Inactive Publication Date: 2009-12-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological diagnosis methods include animal inoculation tests and cell culture methods, which are complex and time-consuming
Serological diagnostic methods include agar diffusion (AGP) test, hemagglutination test, hemagglutination inhibition (HI) test, neuraminidase inhibition (NI) test, enzyme-linked immunosorbent assay (ELISA), virus neutralization test (SN) And immunofluorescence (IF), etc., the shortcoming of serological diagnosis method is that it can only detect the antibody of bird flu virus in the animal body but cannot detect the pathogen
Molecular biology diagnostic methods include PCR, nucleic acid probe technology, etc., which are faster and more sensitive than the first two methods, but require the use of special instruments (such as PCR machines), and the preparation of fluorescent probes is relatively expensive, which is not suitable for grassroots applications

Method used

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  • Subtype H5 avian influenza virus detection kit and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, the preparation of H5 subtype avian influenza virus detection kit

[0058] 1. Synthesis of primers

[0059] Artificially synthesize the following 3 pairs of primers:

[0060] F3: GTGAATTGGAATATGGTAACTGC;

[0061] B3: CCATTCCCTGCCATCCTC;

[0062] FIP: CGATGGTGAGAGGGTGTATGTTTTAAACTCCAATGGGGGCG;

[0063] BIP: CTTGCGACAGGGCTCAGAAATTTTCTGCTATAGCTCCAAATAGTCC;

[0064] LF: TGTGGAATGGCATACTAGAGTT;

[0065] LB: AGCCCTCAAGGAGAGAGA.

[0066] 2. Preparation of LAMP reaction solution

[0067] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025 μL, 0.2 μmol MgSO 4 , 20 μmol betaine (Betaine), 0.035 μmol each of the four deoxynucleotides (dNTPs), 0.04 μmol upstream inner primer (FIP), 0.04 μmol downstream inner primer (BIP), 0.004 μmol upstream outer primer (F3), 0.004 μmol Downstream outer primer (B3), 0.02 μmol upstream circular primer (LF), 0.02 μmol downstream circu...

Embodiment 2

[0070] Embodiment 2, the preparation of H5N1 subtype avian influenza virus detection kit

[0071] 1. Synthesis of primers

[0072] Same as Step 1 of Example 1.

[0073] 2. Preparation of LAMP reaction solution

[0074] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025 μL, 0.2 μmol MgSO 4 , 20 μmol betaine (Betaine), 0.035 μmol each of the four deoxynucleotides (dNTPs), 0.06 μmol upstream inner primer (FIP), 0.06 μmol downstream inner primer (BIP), 0.008 μmol upstream outer primer (F3), 0.008 μmol Downstream outer primer (B3), 0.04 μmol upstream circular primer (LF), 0.04 μmol downstream circular primer (LB), 16 U of Bst DNA polymerase, 0.2 U of AMV reverse transcriptase, sterile double distilled water.

[0075] 3. Assembly of kit

[0076] Same as Step 3 of Example 1.

Embodiment 3

[0077] Embodiment 3, the preparation of H5 subtype avian influenza virus detection kit

[0078] 1. Synthesis of primers

[0079] Same as Step 1 of Example 1.

[0080] 2. Preparation of LAMP reaction solution

[0081] Each 23 μL LAMP reaction solution contains the following components: 0.5 μmol Tris-HCl, 0.25 μmol KCl, 0.25 μmol (NH 4 ) 2 SO 4 , Tween20 0.025 μL, 0.2 μmol MgSO 4 , 20 μmol betaine (Betaine), 0.035 μmol each of the four deoxynucleotides (dNTPs), 0.05 μmol upstream inner primer (FIP), 0.05 μmol downstream inner primer (BIP), 0.006 μmol upstream outer primer (F3), 0.006 μmol Downstream outer primer (B3), 0.03 μmol upstream circular primer (LF), 0.03 μmol downstream circular primer (LB), 16 U of Bst DNA polymerase, 0.2 U of AMV reverse transcriptase, sterile double distilled water.

[0082] 3. Assembly of kit

[0083] Same as Step 3 of Example 1.

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Abstract

The invention discloses a H5 subtype avian influenza virus detection kit and application thereof. The kit provided by the present invention includes three pairs of primers, that is, an inner primer pair, an outer primer pair and a circular primer pair combined with the HA gene of the avian influenza H5N1 virus. The kit also includes a loop-mediated isothermal amplification reagent, a positive control, a negative control, and a fluorescent chromogenic reagent, and the positive control is H5 subtype avian influenza virus RNA. The invention also provides a method for detecting whether an animal carries the H5 subtype avian influenza virus by using the kit of the invention. The H5 subtype avian influenza virus detection kit of the present invention has high detection sensitivity, 6-10 copies can detect the target RNA, is simple and convenient to operate, and is especially suitable for pathogen detection at the grassroots level and H5 that may be contaminated in animal food. Detection of subtypes of avian influenza virus.

Description

technical field [0001] The invention relates to a detection kit for H5 subtype avian influenza virus and its application. Background technique [0002] Avian influenza viruses belong to the Orthomyxoviridae family Influenzavirus type A influenza virus. Avian influenza is an infectious disease characterized by respiratory system and systemic sepsis caused by avian influenza virus infecting poultry. Since December 2003, large-scale avian influenza outbreaks (all of which are highly pathogenic avian influenza H5N1) have broken out in Asian countries such as Vietnam, South Korea, and Japan, which not only caused huge economic losses to the poultry industry in the world, but also brought great harm to human beings. Health and life safety pose a serious threat. According to the statistics of the World Health Organization, as of May 31, 2007, 309 cases of bird flu infection have been confirmed worldwide, and 187 cases have died. [0003] There are many methods for detecting H5 s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 吴文学黄书林
Owner CHINA AGRI UNIV