Preparation and application method for gene chip for detecting drug resistance of A type influenza virus epidemic virus strain

A technique for circulating strains, influenza viruses, applied in the directions of microorganism-based methods, biochemical equipment and methods, determination/testing of microorganisms, etc.

Inactive Publication Date: 2011-11-23
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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Problems solved by technology

However, in one test, it is possible to distinguish whether the influenza virus infected by the patient is one or more of the three subtypes of seasonal H1N1, A-H1N1, and seasonal H3N2, and at the same time detect whether the infected influenza virus is Gene chips resistant to oseltamivir and amantadine have not been reported

Method used

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  • Preparation and application method for gene chip for detecting drug resistance of A type influenza virus epidemic virus strain
  • Preparation and application method for gene chip for detecting drug resistance of A type influenza virus epidemic virus strain
  • Preparation and application method for gene chip for detecting drug resistance of A type influenza virus epidemic virus strain

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Embodiment 1

[0046] Development of gene chip for detection of drug resistance of circulating strains of influenza A virus

[0047] 1. Design of primers

[0048] Log on to the NCBI website, and retrieve the full sequences of the NA and M genes of the three influenza virus subtypes: seasonal H1N1, influenza A H1N1, and seasonal H3N2 in GenBank. Using bioinformatics software, the NA gene and M gene of the three influenza virus subtypes were compared to find the oseltamivir resistance site on the NA gene and the amantadine resistance site on the M gene. The drug resistance loci determined to be detected were: oseltamivir N1 type drug resistance mutation site was NA gene H274Y, N2 type drug resistance mutation site was NA gene E119V; amantadine drug resistance mutation site was M gene V27A and S31N. A number of RT-PCR specific upstream and downstream primers were designed in the upstream and downstream conserved regions of the drug resistance site. After screening, a total of 9 upstream and d...

example 2

[0077] Detection of Throat Swab Samples for Clinical Influenza Virus

[0078] Throat swab culture samples of clinical patients are provided by Zhejiang Yiwu CDC, and the method of the present invention is used for typing detection of the clinical samples.

[0079] 1. Chip preparation

[0080] First, put the centrifuge tube containing the probe (dry powder) on a high-speed centrifuge at 12,000 rpm, centrifuge for 5 minutes (the probe should not be opened before centrifugation), add deionized water to dissolve to a concentration of 100 μM, and use a shaker Collect the liquid on the wall of the tube by quick centrifugation after mixing. After the mixed probe solution was left for 1 hour, 10 μl was taken respectively, mixed with 10 μl spotting buffer and added to a 384-well plate, and each probe was spotted on the aldehyde group using a Pixsys 5000 chip preparation instrument (Cartesian Technologies). On the chemical glass slide, keep a certain humidity during the spotting process...

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Abstract

The invention relates to a preparation and application method for a gene chip for detecting drug resistance of A type influenza virus epidemic virus strains. The gene chip comprises 29 specific oligonucleotide probes, one quality control probe, and nine specific primers and vectors for RT-PCR amplification, which can be used to detect drug resistance of such three subtypes of influenza as seasonal H1N1, A type H1N1 and seasonal H3N2 to oseltamivir and amantadine, wherein all the probes are distributed on the vectors. The invention also includes a preparation method for the gene chip, and the method comprises the following steps: 1, design of primers and probes; 2, synthesis of probes; 3, preparation of the chip. The invention further includes a preparation method for the gene chip, and the method comprises the following steps: 1, extraction of RNA genome of an influenza virus; 2, two sets of RT-PCG amplification; 3, hybridization of chips; 4, cleaning of the chips after hybridization; 5, scanning of the chips; 6, data analysis. The invention has the characteristics of rapidness, accuracy, high flux, high sensitivity, high singularity, etc.

Description

technical field [0001] The invention relates to a method for preparing and using a type A influenza virus epidemic strain drug resistance detection gene chip, which belongs to the field of biochips. Background technique [0002] Influenza virus belongs to Orthomyxoviridae according to virus taxonomy. Its genome is segmented single-stranded negative-sense RNA. Influenza is divided into three types: A (A), B (B) and C (C) according to the antigenic characteristics of the viral nucleoprotein (NP) and membrane protein (MP) and their genetic characteristics. Among them, type A influenza virus can be divided into multiple subtypes according to its surface hemagglutinin (HA) and neuraminidase (NA) protein structure and gene characteristics. So far, type A influenza virus has discovered 16 hemagglutinins Subtype (H1-16), neuraminidase has 9 subtypes (N1-9). Since the influenza virus genome RNA is segmented, gene reassortment between different strains of the same type is prone to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C40B50/06C12R1/93
Inventor 王升启张英杰陈苏红刘琪琦
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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