A Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for feline coronavirus

BACKGROUND Loop-mediated isothermal amplification (LAMP) is a gene amplification technique that amplifies DNA in only one hour and under isothermal conditions, using a DNA polymerase with strand displacement activityand six primers targeting eight regions of the template sequence. AIM OF THE WORK The aim of this study was to develop a reverse transcription loop-mediated isothermal amplification assay for a rapid and inexpensive detection of feline coronavirus (FCoV). METHODS Six primers binding the 3' untranslated region (3'UTR) of the FCoV were designed. Thirty-two samples of RNA from cats (11 feces, 8 effusions, 9 blood samples and 4 tissues) were analyzed and a nested reverse transcription polymerase chain reaction (nRT-PCR) targeting the 3'UTR was also performed. The reaction mixture was incubated in a thermocycler at 63°C for 1 hour followed by 10 minutes at 80°C. LAMP results were evaluated both after electrophoresis migration on agarose gel stained with ethidium bromide and by naked eye after the addition of the hydroxynaphtol blue (HNB) dye. Results were compared with the nRTPCR, considered the gold standard. RESULTS The specificity was 100% on all specimens, while the sensitivity was 100% only on tissues. The overall sensitivity was 52.4% and 50% with gel electrophoresis and HNB, respectively. Discrepant results between the two visualization methods were recorded. DISCUSSION/CONCLUSIONS Except for tissues, the low sensitivity of the LAMP assay for FCoV limits a clinical application of this method. Additional experiments are needed in order to assess the analytical sensitivity and to develop a quantitative visualization technique.