Loop-mediated isothermal amplification assay for the rapid detection of swine influenza virus

Abstract

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at 58 o C and the sensitivity of the RT-LAMP (1 copy/L) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) (10 copy/L) and the same to real time RT-PCR (1 copy/L). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

“…Extracted nucleic acid was stored at -20℃ until further use. RT-LAMP assay for AIV detection was performed using AIV matrix gene-specific primer sets as previously described [ 5 ]. The amplification reaction was performed at 58℃ for 40 min, followed by heating at 80℃ for 5 min to terminate the reaction.…”

“…Because of the high mutation rate of AIV genes, it is difficult to design a multi-set of RT-LAMP primers to detect all subtypes of AIVs [ 10 13 ]. Six primer sets (F3, B3, LF, LB, FIP, and BIP) for the proposed RT-LAMP that specifically target eight different regions highly conserved among all subtypes of AIVs were carefully designed by analyzing most of the AIV matrix gene deposited in the Influenza Sequence Database during 2012–2014 [ 1 5 ]. The RT-LAMP assay using these primers specifically detected all subtypes of AIVs tested, but not HIBV and NDV ( Table 1 ), indicating the assay was highly accurate and specific for all subtypes of AIVs.…”