The development of reverse transcription loop-mediated isothermal amplification for rapid detection of influenza viruses

Abstract

Influenza A infection is a major public health problem world wide. Four major pandemics in the past century including pandemic H1N1 2009 were caused by influenza A viruses. Different approaches of diagnosis assays have been developed to detect influenza A viruses. However, some techniques such as viral isolation, immunofluorescence assay (IFA) and other molecular assays including RT-PCR and real time RT-PCT have limitation to apply in mobile surveillance and unequipped laboratories in developing countries. In this study, one step reverse transcription loop-mediated isothermal amplification was developed as a rapid, sensitive to detect influenza A viruses. To reach the overall goal, a lamp primer set was designed by PrimerExpoler V4 and developed for a sensitive and specific amplification. The optimal amplification reaction is 63oC for 60 minutes then followed by 80oC for ten minutes. The developed assay is ten times more sensitive than conventional RT-PCR and comparable as real time RT-PCR. It is also highly specific for influenza viruses of different hosts. The colorimetric assay of LAMP products is also sensitive as gel electrophoresis. This developed one step RT-LAMP reveals comparable sensitive to detect the Influenza A viruses in both cloacal and tracheal samples. This method is also an easy to use technique and suitable for field surveillance and screening