Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus
Witsanu Rapichai,
Wichayet Saejung,
Kotchaporn Khumtong,
Chaiwat Boonkaewwan,
Supansa Tuanthap,
Peter A. Lieberzeit,
Kiattawee Choowongkomon,
Jatuporn Rattanasrisomporn
Affiliations
Witsanu Rapichai
Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand
Wichayet Saejung
Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
Kotchaporn Khumtong
Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
Chaiwat Boonkaewwan
Akkhraratchakumari Veterinary College, Walailak University, Nakhon Si Thammarat 80161, Thailand
Supansa Tuanthap
Faculty of Veterinary Medicine, Rajamangala University of Technology Tawan-ok, Bangpra, Chonburi 20110, Thailand
Peter A. Lieberzeit
Department of Physical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Strasse 42, A-1090 Vienna, Austria
Kiattawee Choowongkomon
Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand
Jatuporn Rattanasrisomporn
Center for Advanced Studies for Agriculture and Food, Kasetsart University Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand
Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 °C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/µL. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.
