Utility of Reverse Transcription Loop-mediated Isothermal Amplification Assay for Rapid Detection of Novel Influenza Virus (A/H1N1pdm)

Abstract

  A novel influenza virus (A/H1N1pdm) of swine origin emerged in Mexico in early April 2009. The novel influenza virus (A/H1N1pdm) spread rapidly all over the world. In this study, we developed a detection method for this novel influenza virus (A/H1N1pdm) of swine origin based on the reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay. In preliminary examinations, A/H1N1pdm RNA positive control was detected by RT-LAMP assay, RT-LAMP products exhibited a typical ladder pattern, and after digestion with Sau3AI exhibited a single band. The A/Mie/33/2009pdm strain (1.56×10⁶~1.86×10⁶ pfu/mL) was used for analysis of the sensitivities of the RT-LAMP and Real-Time RT-PCR assay. The sensitivity of the RT-LAMP assay was the same as that of the Real-Time RT-PCR assay. The RT-LAMP assay was performed at temperatures of 60, 62.5 and 65℃, and the reaction at 60℃ was the most productive. We detected A/H1N1pdm corresponding to 1.56×10¹~1.86×10¹ pfu/mL by the RT-LAMP assay within 45 min. No cross-reactions of novel A/H1N1pdm with influenza A virus (H1N1), influenza A virus (H3N2), influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, and human boka virus were observed, confirming the specificity of the RT-LAMP assay for detection of A/H1N1pdm. These results indicate that A/H1N1pdm is detected rapidly by the RT-LAMP assay, so this method could become an available diagnostic method suitable for clinical situations requiring quick and appropriate decisions about treatment and care.