Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

• Published in: Molecular and cellular probes Vol. 24; no. 6; pp. 415 - 417
• Main Authors: Cardoso, Tereza C., Ferrari, Heitor F., Bregano, Lívia C., Silva-Frade, Camila, Rosa, Ana Carolina G., Andrade, Alexandre L.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2010
• Subjects: Animals; Coloring Agents; Coronavirus, Turkey - genetics; Cross-reaction; Feces - virology; Hydroxynaphthol blue; LAMP assay; Naphthalenesulfonates - chemistry; Organ Specificity; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; Sensitivity and Specificity; Short Communication; Spike gene; Temperature; Turkey coronavirus; Turkeys - virology; Turkey coronavirus; LAMP assay; Spike gene; Hydroxynaphthol blue
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2010.08.003

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 2 EID 50/50 μl TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum–caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.