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

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.

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus.

Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

Odorant receptor gene expression changes during the parr-smolt transformation in Atlantic salmon.

The ability of salmon to home accurately to their natal stream to spawn has long intrigued biologists and has important consequences for the maintenance of population structure in these species. It is known that olfaction is crucial to homing, and that the transition from the freshwater to the marine environment (the parr-smolt transformation; PST) is a period of increased olfactory sensitivity and learning, resulting in a permanent memory of natal site odours that is retained, at least in part, in peripheral sensory neurones. These odours are then used as cues by sexually maturing fish on their homeward migration. We used quantitative polymerase chain reaction techniques to demonstrate transient increases in expression of odorant receptor transcripts (of up to fifty-fold over pre-PST levels) coincident with PST. Both olfactory (SORB) and vomeronasal receptors (SVRA and SVRC) are involved, which suggests that the fish learn both environmental odours and semiochemicals (pheromones). Receptor expression varies between families and changes over time indicating both genetic differences in odour stimuli and multiple periods of olfactory sensitivity. We suggest that changes in OR gene expression may have a role in homing behaviour and thus the maintenance of population structure in Atlantic salmon.