Sensitive Detection of Influenza A (H1N1) Virus by Isothermal Amplification in A Single Tube

A single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of influenza A (H1N1) virus. At first, two sets of primers were designed based on the conserved regions of both M gene and HA gene for the screening of influenza A virus and for the identification of influenza A (H1N1) subtype. Optimization of RT-LAMP reaction at various conditions was then carried out by using the above primers. Positive reactions can be readily observed by a visual inspection based on the turbidity or SYBR Green I fluorescence. The detection limit of this method is 10 RNA copies per reaction, which is higher than that of the Centers for Disease Control and Prevention (CDC) TaqMan assay. To investigate the feasibility of the RT-LAMP assay, 30 clinical specimens were tested, and the sensitivity and specificity were 92% and 100%, respectively, in comparison with the CDC TaqMan assay. Therefore, the RT-LAMP assay proposed is a perspective tool for the rapid screening of influenza A (H1N1) virus at resource-limited areas.