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 infections. Although polymerase chain reaction (PCR)-based methods are currently the mostcommonly used tools for IAV detection, PCR is not ideal for point-of-care testing. In thisstudy, we aimed to develop a more rapid and sensitive method than PCR-based tools todetect IAV using loop-mediated isothermal amplification (LAMP) technology. We designedreverse-transcriptional (RT)-LAMP primers targeting the hemagglutinin gene. RNAs fromreference H1N1 and H3N2 showed specific RT-LAMP signals with the designed primers.We optimized the reaction conditions and developed universal reaction conditions for bothLAMP assays. Under these conditions, the detection limit was 50 copies for both RT-LAMPassays. 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, apositive signal was detected within 25 min after starting the reaction. In conclusion, ourRT-LAMP assay has high sensitivity and specificity for the detection of the H1 and H3 subtypes, making it suitable for point-of-care IAV testing.