Establishment of a rapid method for detection of influenza A virus by a reverse transcriptase loop-mediated isothermal amplification

Objective A reverse transcriptase loop-mediated isothermal amplification(RT-LAMP)assay was developed for specific,sensitive,rapid,visible detection of influenza A virus. Methods Based on the M gene sequences o f influenza A virus available in GenBank,five primers specific to the eight sites of M gene were designed using PrimerExplorer V4 software,the detection mastermix and reaction conditions were optimized,and the specificity and sensitivity were verified. Results Influenza A virus of H1,H3,H5,H6,H7 and H9subtypes were detected and there was no cross-reaction with influenza B virus,parainfluenza virus,respiratory syncytial virus,human coronavirus,human metapneumovirus,adenoviruses and human bocavirus.The detection limit of RT-LAMP assay was 50copies/μl.Also,the amplification could be finished within 40 min and with the addition of calcein,the presence of influenza A virus could be recognized by naked eyes. Conclusions The newly developed RT-LAMP assay is a specific and simple method for rapid detection of influenza A virus.