Graphical abstract Highlights • Multiplex RT-LAMP for HA and M genes was developed for subtyping influenza A virus. • Multiplex RT-LAMP amplicons were simply analyzed by the colorimetric ICS detection. • Multiplex RT-LAMP (40 min) and ICS detection (15 min) could be complete in 55 min. • Detection sensitivity for the multiplex RT-LAMP and ICS was 10 copies of viral RNA. • Our methodology provides simple, rapid genetic analysis platform for viral detection. Considering the fatal human victims and economic loss by the annual epidemic influenza virus, the development of a rapid and convenient genetic analysis methodology is demanding for timely on-site pathogen detection. In this study, we utilized reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for multiplex target gene amplification, and the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus. Through the optimized primer design, reaction temperature and time, and concentration of enzymes ( Bst DNA polymerase and AMV reverse transcriptase) and dNTP, the HA (H1, H3, and H5 gene) and conserved M gene were amplified. The ICS contains two test lines in addition to a control line in order to detect the presence of the HA and M gene, thereby informing us of influenza virus A type as well as its subtype (H1N1, H3N2, and H5N1). The combination of the multiplex RT-LAMP with the ICS could be complete in 40 min and the pathotyping and subtyping of influenza A virus were performed even with 10 copies of viral RNA templates. Moreover, the subtyping of clinical samples, which were obtained from patients infected by influenza A virus was successfully confirmed using the multiplex RT-LAMP and ICS techniques, showing great feasibility of our methodology for real sample analysis with high speed, simplicity and sensitivity.
【저자키워드】 influenza A virus, Subtyping, Multiplex reverse transcription loop-mediated isothermal amplification, Immunochromatographic strip,