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Primers for detecting influenza virus by RT-PCR and method for detecting influenza virus

An RT-PCR, influenza virus technology, applied in the field of RT-PCR detection technology, can solve the problems of time-consuming operation and high laboratory requirements, and achieve the effect of convenient application and simple operation

Inactive Publication Date: 2009-10-21
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The operation is time-consuming and takes 3 weeks, and the requirements for the laboratory are relatively high

Method used

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  • Primers for detecting influenza virus by RT-PCR and method for detecting influenza virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Design and synthesis of influenza A virus H1N1RT-PCR oligonucleotide primers:

[0044] Download swine-origin influenza virus H1N1 and influenza A viruses H1N1, H3N2, H5N1 and other subtypes M gene and HA gene complete sequence, the software compares and analyzes the consistency of the M gene sequence of all influenza A viruses, and selects a relatively conserved region to design primers; swine H1N1, new type A H1N1 and seasonal influenza A All HA gene sequences of the virus H1N1 were compared together, and primers were selected for the conserved region and the variable region respectively. In primer design, 2 or less degenerate bases are allowed at the same variable site. Screen the extracted candidate primers that meet the following requirements: ①The probe length L is between 19~28bp; ②Tm value is between 42~59℃; ③GC% is between 25~75%; ④polyN≤4bp; ⑤Hairpin ≤4bp; ⑥ coverage >90%; ⑦ BLAST screening, specificity score > L×0.4. The optimal Tm value is se...

Embodiment 2

[0045] Embodiment two: the present invention detects the application example of unknown virus:

[0046] 1. Extraction of viral RNA:

[0047] Take 200 μL of virus sampling solution, add 500 μL of lysis solution, and extract 50 μL of viral RNA according to the instructions of RNeasy Mini Kit (Qiagen, catalog #74104).

[0048] 2. RT-PCR reaction:

[0049] 1) System configuration: use QIAGEN One step RT-PCR Kit (catalog#210212) reaction solution.

[0050]

[0051] 2) RT-PCR: put the reaction tube with the above reaction system in the PCR machine for RT-PCR, the reaction procedure is as follows

[0052] 60°C for 1 minute;

[0053] 42°C for 10 minutes;

[0054] 50°C for 30 minutes;

[0055] 95°C for 15 minutes;

[0056] Denaturation at 94°C for 30 seconds;

[0057] Anneal at 50°C for 30 seconds;

[0058]Extend at 72°C for 1 minute;

[0059] Go back to step 5, 34 cycles;

[0060] 72°C for 10 minutes;

[0061] Store at 4°C.

[0062] 3. Detection of RT-PCR products: Take...

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Abstract

The invention discloses primers for detecting influenza virus by RT-PCR and a method for detecting the influenza virus. The primers comprise three pairs of total six oligonucleotide primer sequences such as influenza A virus generality primer, influenza A H1N1 virus H1 generality primer, novel influenza A H1N1 virus H1 specificity primer and the like; and the invention simultaneously discloses treatment of a sample to be detected, an RT-PCR reaction system and reaction condition, and result analysis. The invention can quickly and effectively determine influenza A virus, influenza A H1N1 virus and novel influenza A H1N1 virus, and provide feasible technical support for influenza epidemic early warning mechanisms in the fields such as clinical diagnosis, inspection and quarantine and the like.

Description

technical field [0001] The invention relates to a RT-PCR detection technology, in particular to an influenza virus RT-PCR detection primer and a method for detecting influenza virus. Background technique [0002] The influenza virus genome consists of 8 negative-strand RNA segments. According to nucleoprotein (NP) and matrix protein (M), it is divided into A type, B type and C type. 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] In March 2009, cases of Influenza A (H1N1) virus infection were discovered successively in Mexico and the United States. As of May 21, 2009, confirmed cases of Influenza A (H1N1) virus had appeared in 42 countries, and the number of confirmed cases w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 王伟舒跃龙温乐英李晓丹高荣保王大燕李德新
Owner 中国疾病预防控制中心病毒病预防控制所
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