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

• Published in: Journal of applied microbiology Vol. 109; no. 1; pp. 180 - 189
• Main Authors: Pierce, K.E, Mistry, R, Reid, S.M, Bharya, S, Dukes, J.P, Hartshorn, C, King, D.P, Wangh, L.J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.07.2010; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Amplification; Animals; armored RNA; Assaying; asymmetric PCR; Base Sequence; Biological and medical sciences; Complementary DNA; Deoxyribonucleic acid; Design optimization; diagnosis; DNA; DNA Primers - genetics; Fluorescence; Foot & mouth disease; foot-and-mouth disease; Foot-and-Mouth Disease - diagnosis; Foot-and-Mouth Disease - virology; Foot-and-mouth disease virus; Foot-and-Mouth Disease Virus - genetics; Foot-and-Mouth Disease Virus - isolation & purification; Fundamental and applied biological sciences. Psychology; LATE-PCR; Microbiology; Nucleic Acid Probes - genetics; Nucleotide sequence; Pilot Projects; Polymerase chain reaction; Portable equipment; real-time RT-PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse transcription; Ribonucleic acid; RNA; RNA viruses; RNA, Viral - isolation & purification; Sensitivity and Specificity; Signal processing; Target detection; Viruses; Foot and mouth disease; RNA viruses; foot-and-mouth disease; RNA; Applied microbiology; Reverse transcription; asymmetric PCR; Real time; real-time RT-PCR; Optimization; Infection; Design; Polymerase chain reaction; Virus; armored RNA; Analysis method; Viral disease; Diagnosis; Detection; Reverse transcription polymerase chain reaction; LATE-PCR
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2009.04640.x

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). 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. 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. 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.