Development of reverse transcription loop-mediated isothermal amplification assays for point-of-care testing of avian influenza virus subtype H5 and H9

摘要

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in additionto economic losses to the poultry industry. In this study, we aimed to develop a rapid andsensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP)technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMPtargeting the universal M gene was designed to screen for the presence of AIV and RTLAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5and H9 subtypes. All three RT-LAMP assays showed specific amplification results withoutnonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assayswere 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than thedetection limits of the reference reverse-transcription polymerase chain reaction (RT-PCR)(1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays wasless than 30 min, which was approximately four times quicker than that of conventionalRT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.