Development of reverse-transcription loop-mediated isothermal amplification assays for point-of-care testing of human influenza virus subtypes H1N1 and H3N2

Abstract

Influenza A virus (IAV) is the most widespread pathogen causing human respiratory infec-tions. Although polymerase chain reaction (PCR)–based methods are currently the most commonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In this study, we aimed to develop a more rapid and sensitive method than PCR-based tools to detect IAV using loop-mediated isothermal amplification (LAMP) technology. We designed reverse-transcriptional (RT)–LAMP primers targeting the hemagglutinin gene. RNAs from reference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers. We optimized the reaction conditions and developed universal reaction conditions for both LAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMP assays. There was no non-specific signal to 19 non-IAV respiratory viruses, such as influenza B virus, coronaviruses, and respiratory syncytial viruses. Regarding the reaction time, a positive signal was detected within 25 min after starting the reaction. In conclusion, our RT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 sub-types, making it suitable for point-of-care IAV testing.