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Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus

A technology for detecting influenza virus and primers, which is applied in the field of rRT-PCR detection, 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 and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus
  • Primers and probes for detecting influenza virus by rRT-PCR and method for detecting influenza virus

Examples

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Effect test

Embodiment 1

[0048] Example 1: Design and synthesis of influenza A virus H1N1rRT-PCR oligonucleotide primers

[0049] Download swine-origin influenza virus H1N1 and influenza A viruses H1N1, H3N2, The complete sequence of M gene, NP gene and HA gene of H5N1 and other subtypes, the software compares and analyzes the consistency of M gene sequences of all influenza A viruses, and selects relatively conserved regions to design primers and probes; All NP gene and HA gene sequences of seasonal influenza A virus H1N1 were compared together, and relatively conserved regions were selected to design primers and probes. In the design of primers and probes, 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 valu...

Embodiment 2

[0050] Embodiment two: the present invention detects the application example of unknown virus

[0051] 1. Extraction of viral RNA:

[0052] 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).

[0053] 2. rRT-PCR reaction

[0054]1) System configuration: use QIAGEN QuantiTectTM Probe rRT-PCR Kit (catalog#20443) reaction solution

[0055]

[0056] 2) rRT-PCR: put the reaction tube with the above reaction system in the PCR machine for rRT-PCR, the reaction procedure is as follows

[0057] 60°C for 5 minutes;

[0058] 50°C for 30 minutes;

[0059] 95°C for 15 minutes;

[0060] Denaturation at 94°C for 15 seconds;

[0061] Anneal at 50°C for 30 seconds;

[0062] Extend at 72°C for 30 seconds;

[0063] Go back to step 5 for 45 cycles.

[0064] 3. Result judgment:

[0065] The result is judged when the negative and positive control results are estab...

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Abstract

The invention discloses primers and probes for detecting influenza virus by rRT-PCR and a method for detecting the influenza virus. The primers and the probes comprise influenza A virus specificity primer and probe, two groups of novel influenza A H1N1 virus H1 specificity primers and probes, novel influenza A virus specificity primer and probe and the like, namely four kinds of total 12 oligonucleotide primer and probe sequences; and the invention simultaneously discloses treatment of a sample to be detected, a rRT-PCR reaction system and reaction condition, and result analysis. The invention can quickly and effectively determine influenza A virus, novel influenza A H1N1 virus and novel influenza A virus. The primers and the probes and the method have simple operation and convenient application, 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 an rRT-PCR detection technology, in particular to a primer and a probe for rRT-PCR detection of influenza virus 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 around the worl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 高荣保舒跃龙温乐英李晓丹王大燕王伟李德新
Owner 中国疾病预防控制中心病毒病预防控制所
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