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Virology Journal
Published in: Virology Journal 1/2011

Open Access 01-12-2011 | Methodology

Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay

Authors: Yi Peng, Zhixun Xie, Jiabo Liu, Yaoshan Pang, Xianwen Deng, Zhiqin Xie, Liji Xie, Qing Fan, Jiaxun Feng, Mazhar I Khan

Published in: Virology Journal | Issue 1/2011

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Abstract

Background

Recent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment.

Results

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation.

Conclusions

The newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions.
Appendix
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Metadata
Title
Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay
Authors
Yi Peng
Zhixun Xie
Jiabo Liu
Yaoshan Pang
Xianwen Deng
Zhiqin Xie
Liji Xie
Qing Fan
Jiaxun Feng
Mazhar I Khan
Publication date
01-12-2011
Publisher
BioMed Central
Published in
Virology Journal / Issue 1/2011
Electronic ISSN: 1743-422X
DOI
https://doi.org/10.1186/1743-422X-8-337

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