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Kit for detecting influenza A H1N1 viruses and detection method

A technology for detecting kits and influenza viruses, which is applied in biochemical equipment and methods, microbial measurement/testing, fluorescence/phosphorescence, etc. It can solve the problems of long time, few detection sites, and difficulty in finding antibodies, etc.

Inactive Publication Date: 2011-08-03
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method takes a long time and is technically demanding, but it is the most reliable method of diagnosis
(3) Influenza A (H1N1) antigen inspection, which uses immunological technology to check the virus antigen in the specimen with known antibodies, but the virus mutates so quickly that it is difficult to find a matching known antibody
However, they have the following disadvantages: few detection sites, low sensitivity, high false positive rate

Method used

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  • Kit for detecting influenza A H1N1 viruses and detection method
  • Kit for detecting influenza A H1N1 viruses and detection method
  • Kit for detecting influenza A H1N1 viruses and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1 primer, probe, verification template design

[0094] Through the comparison and analysis of the nucleic acid sequences of the HA gene and NP gene of 24 influenza A H1N1 viruses published as of May 4, 2009, their conserved regions were obtained, and all influenza A viruses were analyzed More than 6,000 HA genes and more than 3,000 NP sequences were used to find the specific sites of influenza A (H1N1) virus, and to design real-time fluorescent quantitative PCR primers and TaqMan probes, as well as the sequence of the conserved fragment of the target gene. A total of 2 conserved fragment sequences on the HA gene and 2 conserved fragment sequences on the NP gene, as well as 8 primer sequences and 4 probe sequences were obtained. Application analysis software includes bioinformatics analysis software such as VectorNTI and AlleleID.

[0095] The target gene fragment sequence is as follows ((5'-3')):

[0096] HA-1

[0097] TTGAGCTCAGTGTCATCATTTGAAAGGTTTGAGATAT...

Embodiment 2

[0120] Embodiment 2 real-time PCR system establishment

[0121] Equipment: RocheLightCycler real-time fluorescent quantitative PCR instrument.

[0122] Reagents: primers, positive control template (equal molar mixture of pGMT-HA and pGMT-NP plasmids) Synthesis: Beijing Aoke Biotechnology Co., Ltd., PAGE purification. Verify that the template is plasmid DNA.

[0123] Probe synthesis: Treasure Bioengineering (Dalian) Co., Ltd., the 5' end was connected to the 6-Fam group, the 3' end was connected to the BHQ-1 group, and purified by HPLC.

[0124] dNTPMixture, Taq DNA polymerase, Mg 2+ , PCR buffer: Promega (Beijing) Biotechnology Co., Ltd.

[0125] Sterile deionized water: self-made.

[0126] Environment: independent sample processing, reagent configuration, PCR reaction laboratory.

[0127] reaction system:

[0128] Reagent

volume

10×Buffer

2.0 μL

10 mM various dNTPs

0.4μL

25mMMg 2+

3.2μL

10pM upstream primer

...

Embodiment 3

[0134] Embodiment 3 primers, probe combination screening

[0135] Such as figure 1 As shown, through the previous screening experiments on more than 300 combinations of more than 500 primer pairs and probes, 4 sets of combinations can be determined at present, and their sensitivity and specificity indicators meet the expected requirements.

[0136] The 4 sets of combinations are as follows.

[0137]

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Abstract

The invention provides a kit for detecting influenza A H1N1 viruses and detection method. The kit respectively carries out specificity detection on two different zones (HA-1 and HA-2) on HA gene and two different zones (NP-1 and NP-2) on NP gene of the influenza A H1N1 viruses, which detects four loci of the influenza A H1N1 viruses, and reduces false negative rate caused by virus mutations, thereby achieving the purpose of improving detection rate.

Description

technical field [0001] The invention relates to an influenza virus detection kit and a detection method, in particular to a type A (H1N1) influenza virus detection kit and a detection method. Background technique [0002] From the end of March to mid-April 2009, a new type of H1N1 influenza broke out successively in Mexico, the United States and other places. In less than a month, the epidemic has spread to 34 countries or regions around the world, and the number of confirmed infections has reached 6044, of which 63 died (as of May 13, 2009). At present, China has discovered and diagnosed imported influenza A (H1N1) patients. It can be seen that the rapid and sensitive detection technology of influenza A (H1N1) virus has become the first way to protect people's lives and health and prevent and control the epidemic in a large scale in China. barrier. [0003] Influenza viruses belong to the Orthomyxoviridae family. According to the characteristics of the nucleoprotein (NP)...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 王绪敏任鲁风张猛于军朱文斯单广乐王丽
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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