Development and application of a rapid detection system for swine influenza virus by a reverse transcription loop-mediated isothermal amplification assay

Based on the published sequences of different serotypes of swine influenza virus (Sty), six degenerate primers were designed to target the conserved region of the SI V polymerase A (PA) gene. An Sly reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established by employing the genomic material from Sly H1, H3, H5, and H9 subtype strains as templates. Reactions performed using a Loopamp LA-320 turbidimeter demonstrated that the four SIV subtype strains were specifically amplified within 45 min under isothermal conditions at 63 degrees C. This study demonstrated that there was effective amplification of the RNA template diluted 10(4)-fold, and 19 clinical samples were SIV positive in concordance with detection by conventional virus isolation techniques. Further validation by gene chip analysis showed that there was 100% parity compared with RT-LAMP. Of the 79 clinical samples analyzed by RT-LAMP indicating that the assay was suitable for SIV detection and was rapid, specific, and sensitive.