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Flu/human avian influenza virus detection gene chip and production method and use

A technology for detecting gene chips and avian influenza viruses, applied in the field of gene chip detection, can solve the problems of low detection accuracy and long detection time, and achieve the effects of high detection accuracy and short detection time

Inactive Publication Date: 2009-03-25
中国疾病预防控制中心病毒病预防控制所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Long detection time and low detection accuracy

Method used

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  • Flu/human avian influenza virus detection gene chip and production method and use
  • Flu/human avian influenza virus detection gene chip and production method and use
  • Flu/human avian influenza virus detection gene chip and production method and use

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0065] Design and synthesis of oligonucleotide probes for human / avian influenza virus detection:

[0066] Find the H1N1, H3N2, H5N1, and H9N2 subtypes of influenza A viruses from GenBank's Influenza virus resources database (Influenza virus resources, http: / / www.ncbi.nlm.nih.gov / genomes / FLU / FLU.html) The HA gene sequence and NA gene sequence, as well as the A and B type NP gene sequences, are aligned separately to divide the conservative region, allowing 2 or less degenerate bases at the same variable site, within the conservative region Candidate probes were extracted at intervals of 1bp. Screen the extracted candidate probes that meet the following requirements: ① probe length L is between 19 and 28 bp; ② Tm value is between 42 and 59 °C; ③ GC% is between 25 and 75%; ④ polyN≤4 bp; ⑤Hairpin≤4bp; ⑥coverage >90%; ⑦screened by BLAST, specificity score>L×0.4. The optimal Tm value is set at 47°C, and the optimal probe length is 25bp. If there are overlapping probes, the positio...

specific Embodiment 2

[0068] Preparation of Gene Chip for Influenza / Human Avian Influenza Virus Detection:

[0069] Aldehydized glass slides were purchased from Boao Chip Company, and chips were prepared in Boao Chip Company. Each probe was diluted to 40 μmol / L with ddII20 for use. When spotting, the oligonucleotide probe was diluted with 2× spotting solution to a final concentration of 20 μmol / L, and 10 μL was transferred to a 384-well plate. Use a spotting system to spot the probes on the aldehyde chip, with a spot diameter of 160 μm and a spot spacing of 300 μm. After sample application, the chip was placed in a wet box at 37°C for more than 12 hours to hydrate, then the chip was washed with washing solution (0.2% SDS), blocked in blocking solution (NaBH4 solution) for 5 minutes, washed with water, and dried by centrifugation.

specific Embodiment 3

[0070] The present invention detects the application example of unknown virus:

[0071] Step 1, extraction of viral RNA:

[0072] Take 200 μL of virus sampling solution, add 600 μL of lysing solution, and extract viral RNA according to the instructions of RNeasy Mini Kit (Qiagen, catalog #74104).

[0073] Step 2, reverse transcription of viral RNA into cDNA (one strand):

[0074] M-MLV RTase cDNA Synthesis Kit (TaKaRa Company, catalog#D6130) was used for reverse transcription and cDNA first-strand synthesis steps

[0075] 20μL reverse transcription system:

[0076] Viral RNA 13μL

[0077] 5×1st Strand Synthesis Buffer 4μL

[0078] dNTP Mix (10mM each) 1μL

[0079] RNase Inhibitor (20U / μL) 0.1μL

[0080] UF12 (10μM) 1μL

[0081] M-MLV (200U / μL) 1μL

[0082] Reaction conditions: place at room temperature for 10 minutes, react at 42°C for 60 minutes, and ice-bath for 2 minutes.

[0083] Step 3, the synthesis of viral cDNA (two strands):

[0084] reaction system:

[008...

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Abstract

The invention discloses an influenza / human and avian influenza virus detection gene chip and a preparation method and an application thereof, 102 probes used for detecting A type, B type, H1N1 hipotype, H3N2 hipotype, H5N1 hipotype and H9N2 hipotype influenza viruses and 4 highly pathogenic differential diagnosis probes, as well as a process of sample treatment, hybridization, elution and chip identification which can carry out detection, typing or identification over the 6 types of viruses simultaneously and can identify the high pathogenicity of viruses. The invention not only greatly shortens the detection time of influenza viruses, but also improves the detection accuracy and provides a feasible technique support for the early warning mechanism of influenza epidemic situation in fields of clinical diagnosis, inspection quarantine, and the like.

Description

technical field [0001] The invention relates to a gene chip detection technology, in particular to a gene chip for detection of influenza / human avian influenza virus and its production method and application. Background technique [0002] The influenza virus genome is composed of 8 negative-strand RNA segments, which are divided into A, B, and C types according to the nucleoprotein (NP) and matrix protein (M). Among them, type A influenza virus is divided into 16 HA subtypes and 9 NA subtypes according to the difference of surface hemagglutinin (HA) and neuraminidase (NA); type B and type C influenza viruses are not divided into subtypes. Type A often causes a worldwide pandemic; Type B and Type C are characterized by local outbreaks and sporadic epidemics. [0003] my country is a place where influenza frequently occurs. H1N1 subtype, H3N2 subtype and B influenza virus are the main seasonal influenza virus epidemic strains in my country at present, and H1N1 subtype is also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 贾菲高荣保舒跃龙刘宏生
Owner 中国疾病预防控制中心病毒病预防控制所
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