Detection of influenza A virus by reverse transcription-loop mediated isothermal amplification

Abstract

Objective To detect influenza A virus by reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to established a rapid, simple and visualization nucleic acid detection method. Methods The RT-LAMP primers were designed in accordance with the hemagglutinin gene of influenza A virus. Then, the specificity of the primers was evaluated by detection of different influenza viruses, and the sensitivity was confirmed by testing multiple diluted RNA samples. Hydroxynaphthol blue (HNB) was used for visually evaluation and gel electrophoresis was used for validation. Clinical samples were detected by RT-LAMP assay. Its consistency with fluorescent quantitative polymerase chain reaction (PCR) methods was compared. Results The primers of RT-LAMP assay had high specificity. This technique could amplify influenza A virus accurately. The detection limit of RT-LAMP assay was 2.5×103 copies/mL by detection of multiple diluted RNA samples. In addition, the results of RT-LAMP assay could be visually inspected using HNB by color change, and the results was in accordance with that of gel electrophoresis. RT-LAMP assay was in consistence with fluorescent quantitative PCR when clinically applied. Conclusions RT-LAMP assay is a rapid, specific, sensitive and simple method to detect influenza A virus. Key words: Influenza A virus; Reverse transcription-loop mediated isothermal amplification; Rapid detection