Development of one-step reverse transcription loop-mediated isothermal amplification (LAMP) as a screening tool for influenza A viruses

Abstract

Influenza A virus is a major cause of influenza pandemics and can infect several host species including humans and animals. The objective of this study was to develop a one-step reverse transcription loop-mediated isothermal amplification assay (LAMP) for the detection of genetically diverse influenza A viruses from both human and anim al hosts. First, a set of two inner and two outer primers were designed based on the conserved region of the matrix (M) gene of influenza A viruses. The amplification reaction was optimized at 63oC for 60 min and performed in a simple heat block. The amplicons could be visualized either by gel electrophoresis or by visual analysis upon addition of SybrGreen. The developed LAMP assay was tested with 50 influenza A isolates including H1N1, H1N2, H3N2, H5N1 and H7N4 from swine, avian and human hosts. In sensitivity test, the assay detection capability was ten times more sensitive than conventional RT-PCR and comparable to real time RT-PCR. In summary, this assay is a rapid, simple and sensitive assay suitable for less-equipped laboratories and thus can be utilized in the field as a screening test.