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Gene chip for detecting influenza A virus and preparation method and application thereof

An influenza virus and gene chip technology, applied in the field of genetic engineering, achieves broad application prospects, overcomes time-consuming and laborious effects, and overcomes the effects of a single detection method

Inactive Publication Date: 2010-05-05
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] According to the relevant literature retrieved comprehensively, most of the studies on influenza virus typing and identification only involve some subtypes of influenza virus, and the methods used include cDNA chip, oligonucleotide probe chip and other technologies. Identification with NA subtypes has not been reported

Method used

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  • Gene chip for detecting influenza A virus and preparation method and application thereof
  • Gene chip for detecting influenza A virus and preparation method and application thereof
  • Gene chip for detecting influenza A virus and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] One: Design and synthesis of probes and primers for detecting influenza A virus

[0048] Log in to NCBI to search GenBank for the full M gene of influenza A virus, the full HA gene of 1-16 subtype A influenza virus, and the full NA gene of 1-9 subtype influenza A virus. With the help of bioinformatics software, design multiple sets of specific primers for M genes, 1-16 subtype A influenza virus HA genes, and 1-9 subtype A influenza virus NA genes, and establish an M RT-PCR detection method. Design multiple specific oligonucleotide probes. In the probe, the DNA fragment selected from the nucleotide sequence of the M gene of the influenza A virus has the base sequence shown in SEQ ID NO: 1-4; from H1-16 subtype influenza A viruses The DNA fragment selected from the nucleotide sequence of the HA gene has the base sequence shown in SEQ ID NO: 5-31; the DNA is selected from the nucleotide sequence of the NA gene of the N1-9 subtype A influenza virus The fragment has the base ...

experiment example 1

[0176] Using the gene chip prepared by the present invention to detect 6 specimens of clinically suspected influenza A H1N1 virus from Jilin Province CDC

[0177] One: chip spotting

[0178] The synthesized H1, H2, H3, N1, N2 subtype and M probe dry powder tube are first dissolved. The steps are as follows: 12000 rpm, centrifugation for 5 minutes (be careful not to open the lid of the dry powder tube before centrifugation), add double-distilled water, dissolve to a concentration of 60μM, homogenize with a shaker stick and quickly centrifuge to remove the liquid on the tube wall Leave. After standing at room temperature for 1 hour, add the probe to the corresponding position in the 384-well plate, add 10 μL of probe to each well and 10 μL of DMSO. Aldehyde-based glass slides were purchased from Boao Chip Company, and the probes in the 384-well plate were spotted on the aldehyde-based glass slides using an automatic spotting instrument to form a microarray designed by us (see Figu...

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Abstract

The invention relates to a gene chip for typing influenza A virus, a preparation method thereof, a detection method and a detection kit, wherein the gene chip comprises a solid phase carrier and an oligonucleotide probe which is fixed on the solid phase carrier and contains DNA segments selected from one or more genes of HA, NA and / or M of the influenza A virus. The gene chip of the invention has very high sensitivity and specificity, can be used for simultaneously detecting a plurality of groups of influenza A virus genes and identifying groups by adopting a multiple PCR amplification mode, has wide application prospects, can identify the specific serotype, has efficient and quick detection and analysis methods, and can be applied to the fields of life science, medical testing, food safety, environmental surveillance, import and export inspection and quarantine, bioterrorism prevention, epidemiological surveillance, and the like.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, in particular to a gene chip for detecting multiple A influenza viruses, a preparation method and application thereof. Background technique [0002] Influenza is an acute respiratory infection caused by influenza virus, and it is also a highly contagious and fast spreading disease. The influenza virus genome contains 8 single-stranded negative-strand RNA virus segments, which are currently divided into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. The subtype identification mainly relies on hemagglutination inhibition test and Neuraminidase inhibition test, which is a test method commonly used by the World Health Organization (WHO) for global influenza surveillance. [0003] Gene chip technology is a rapid, parallel, and high-throughput genetic-level diagnostic technology that can be applied to parallel detection of hundreds of disease-related molecules. Its emergence provides the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12N15/11C12R1/93
Inventor 金宁一田明尧鲁会军李昌李霄金扩世南文龙谭磊张金双赵翠青孙珊珊颜雯
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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