Abstract

Conventional reverse transcriptase polymerase chain reaction (RT-PCR) and optimized of a closed tube reverse-transcription loop-mediated isothermal amplification (RT-LAMP) were used for detection of pandemic (H1N1) 2009 virus and the optimized of a closed tube RT-LAMP methods were compared with the conventional RT-PCR with respect to specificity and sensitivity. In this study, optimized RT-LAMP detected 2 copies of target RNA by visual detection with modified dye. Reaction time, temperature and quantity of each reagent were optimised for the detection of the virus. The sensitivity of detection limit by optimised RT-LAMP was 100 times as that of conventional RT-PCR. Amplification of DNA can be identified by visualization with modified dye, which reduces the cross-contamination caused by opening tube. The sensitivity of visual detection was equivalent to that of electrophoresis analysis. Additionally, the method was specific as no cross-reaction was observed among samples from human blood, Escherichia coli and other related viruses including human seasonal influenza A, subtypes H1N1, H1N2 and H3N2 viruses. These results demonstrate that the optimized RT-LAMP assay for pandemic (H1N1) 2009 virus RNA was a valuable tool with simplicity, rapidity and specificity, as well as its superiority for the screening and surveillance of influenza in developing countries.

Key words: Pandemic (H1N1) 2009 virus, loop-mediated isothermal amplification (LAMP), reverse transcriptase polymerase chain reaction (RT-PCR), HA gene.

Abbreviation

HA, Hemagglutinin; NP, neuraminidase; NS, non-structural proteins; PB, polymerase basic protein; PA, polymerase acidic protein; RT-PCR, reverse transcription polymerase chain reaction; LAMP, loop-mediated isothermal amplification; DTT,dithiothreitol; PPV, positive predictive value; NPV, negative predictive value.