Design and optimization of a novel reverse transcription linear‐after‐the‐exponential PCR for the detection of foot‐and‐mouth disease virus

Abstract

Aims: A novel molecular assay for the detection of foot‐and‐mouth disease virus (FMDV) was developed using linear‐after‐the‐exponential polymerase chain reaction (LATE‐PCR). Methods and Results: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE‐PCR to quantify initial target concentration through endpoint detection. A two‐step protocol involving reverse transcription (RT) followed by LATE‐PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE‐PCR were combined in a one‐step duplex assay for co‐amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA^®. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre‐incubation, then generate cDNA strands during a 3‐min RT step at 60°C, and the resulting cDNA is amplified by LATE‐PCR without intervening sample processing. Conclusions: The RT‐LATE‐PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch‐tolerant probe. Significance and Impact of the Study: In addition to offering improvements over current laboratory‐based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable (‘point‐of‐care’) device, the BioSeeq^®II, designed for the rapid diagnosis of FMD in the field.