Department of Health, Animal Science and Food Safety (VESPA)
Doi
Abstract
Loop-mediated isothermal amplification (LAMP) is a molecular method that amplifies DNA underisothermal conditions. It relies on the use of 4 different primers recognizing 6 regions of the templatesequence and on the use of a DNA polymerase with strand displacement activity (Notomi et al., 2000).The addition of two loop primers allows the reaction time to be of one hour only (Nagamine et al., 2002).The aim of this study was to develop a reverse transcription LAMP assay for an easy and inexpensivedetection of feline Coronavirus (FCoV). Six primers binding the conserved 3’UTR region of the FCoVwere designed with the Primer Explorer software. Thirty-two samples of RNA (11 feces, 8 effusions, 9blood samples and 4 tissues) on which a reverse transcription polymerase chain reaction (RT-PCR) forthe 3’UTR region was performed were used. The reaction was carried out in 25μL reaction volume andthe mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMPproducts were visualized under UV after electrophoresis migration on a 1.5% agarose gel stained withethidium bromide, where they produce a ladder-like pattern if positive. Results where compared withthose obtained on standard PCR. Sensitivity and specificity were respectively 60% and 100% on feces,40% and 100% on effusions, 25% and 100% on blood, and 100% and 100% on tissues. The overall sensitivityand specificity of this method were of 57.1% and 100%, thus limiting a clinical application of this method,except for tissues
