Simultaneous detection of new H1N1 A influenza virus and human seasonal H1N1 and H3N2 influenza viruses by multi-fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR)

A multiplex fluorescent quantitative and RT-PCR technology, applied in the field of biotechnology applications, can solve problems such as dependence on equipment, low sensitivity, and inability to detect new influenza A (H1N1) viruses

Inactive Publication Date: 2011-06-08
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Early studies on influenza virus typing used methods such as fluorescent quantitative RT-PCR, chip, flow cytometry and sequencing as the terminal detection system, but most of these methods cannot detect the new type A H1N1 influenza virus
Although some methods have been further developed, they still rely on expensive equipment and are not very sensitive
Four fluorescent quantitative RT-PCR techniques have been developed, including HPA(H1)v (UK Health Protection Agency), CDC(H1)v (U.S. Centers for Disease Control and Prevention), HPA(N1)v (UK Health Protection Agency) and S -OIV (National Virus Reference Laboratory, Ireland) can detect the new H1N1 influenza virus and seasonal influenza virus, but these methods are multi-tube multiplex fluorescent quantitative RT-PCR, which is cumbersome to operate, takes a long time, and costs more high
Fluorescence quantitative RT-PCR reagents for detection of new influenza A developed by domestic Daan Gene Company and Kehua Company have been approved by the Chinese FDA, but these methods do not include the detection of seasonal H3N2, and cannot simultaneously detect two species in a single tube Seasonal Influenza and Novel Influenza A

Method used

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  • Simultaneous detection of new H1N1 A influenza virus and human seasonal H1N1 and H3N2 influenza viruses by multi-fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR)

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Experimental program
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Effect test

Embodiment approach 1

[0014] Embodiment 1: Reaction system and conditions of multiplex fluorescent quantitative RT-PCR

[0015] Influenza virus RNA from different sources and subtypes was extracted according to Qiagen kit instructions. Establish a Bio-Rad fluorescent quantitative RT-PCR one-step reaction system (25 μl), the composition is as follows: RTase 0.5 μl, itaq mix 12.5 μl, itaq enzyme 0.5 μl, RNA template 4 μl, primers and probes for sH1N1, sH3N2, nH1N1 and RnasP The concentrations were 1.6μmol / L, 1.6μmol / L; 0.2μmol / L, 0.2μmol / L; 0.4μmol / L, 0.4μmol / L; 0.08μmol / L, 0.08μmol / L. The reaction conditions were as follows: cDNA was synthesized at 50°C for 30 minutes, and then iScript reverse transcriptase was inactivated at 95°C for 5 minutes. 50 cycles of PCR included 95°C for 15s, 60°C for 30s, and finally the plate was read with CFX96 Real-Time System (Bio-Rad).

Embodiment approach 2

[0016] Embodiment 2: Specificity of multiplex quantitative RT-PCR

[0017] Human seasonal H1N1, H3N2, HB and A / California / 07 / 2009(H1N1)sw1 cells were used to culture virus strains, and A / California / 07 / 2009(H1N1)sw1 was artificially mixed with HB, sH1N1, The mixed sample of sH3N2 was used as the detection object, and the specificity analysis of multiple fluorescent quantitative RT-PCR was performed after RNA was extracted. There was no cross-reaction, only specific signal appeared in each reaction, and the test result of HB was negative.

Embodiment approach 3

[0018] Embodiment 3: Sensitivity of multiplex quantitative RT-PCR

[0019] The purified and quantified in vitro transcribed human influenza A H1N1 influenza HA whole gene RNA was serially diluted to 2, 20, 200 and 2000 copies, and the system for multiplex fluorescent quantitative RT-PCR detection was the same as in Embodiment 1. The detection sensitivity can reach the level of 20 copies of RNA.

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Abstract

The invention belongs to the field of application of biotechnology, and relates to simultaneous detecting and monitoring of HA genes in human new H1N1 A influenza viruses and human seasonal H1N1 and H3N2 influenza viruses of specimens such as nasopharyngeal swabs of fever patients and the like by disease prevention and control mechanisms of all levels, influenza detection network laboratories, sentinel point hospitals and the like. Conserved sequences of HA genes in human new H1N1 A influenza viruses and human seasonal H1N1 and H3N2 influenza viruses are specifically analyzed by computer software, two corresponding primers and probes are designed respectively, single tube multi (quadruple)-fluorescence quantitative RT-PCR (comprising internal reference genes) is performed, and an entire reaction lasts for no more than 2 hours. The defects of complex operation, long time and high cost existing in the conventional multi-tube multi-fluorescence quantitative RT-PCR method are overcome, a good tool for tracing possible coinfection and recombination of new H1N1 A influenza and seasonal influenza is provided, and powerful technical support is provided for quick and correct screening of a large number of influenza suspected cases by the characteristics of high specificity, high sensitivity and high speed.

Description

field of invention [0001] The invention belongs to the application field of biotechnology. The invention is based on the multiple fluorescence quantitative RT-PCR technology to simultaneously detect the HA gene of human new type A H1N1 and human seasonal H1N1 and H3N2 influenza virus. It is used for simultaneous detection and monitoring of new human influenza A H1N1 and human seasonal H1N1 and H3N2 influenza viruses by disease prevention and control institutions at all levels, influenza detection network laboratories, and sentinel hospitals. Background of the invention [0002] The new type A H1N1 influenza (formerly known as human-infected swine influenza virus) is an acute respiratory infectious disease caused by a new type A H1N1 virus. It is highly contagious and can be transmitted through close-range droplets and contact. The virus was first detected in humans in the United States in April 2009. Subsequently, the virus spread around the world. Sequence studies have s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 马学军秦萌舒跃龙董小平李德新聂凯王淼
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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