title: Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye.
abstract: 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.
authors: UNESP - São Paulo State University, Laboratory of Animal Virology, Campus de Araçatuba, São Paulo, Brazil. tcardoso@fmva.unesp.br
authors: Tereza C
authors: TC
authors: Cardoso
authors: Heitor F
authors: HF
authors: Ferrari
authors: Lívia C
authors: LC
authors: Bregano
authors: Camila
authors: C
authors: Silva-Frade
authors: Ana Carolina G
authors: AC
authors: Rosa
authors: Alexandre L
authors: AL
authors: Andrade
mesh_terms: D000818:Animals; D004396:Coloring Agents; D004752:Coronavirus, Turkey / Q000235:genetics*; D005243:Feces / Q000821:virology*; D009282:Naphthalenesulfonates / Q000737:chemistry*; D009928:Organ Specificity; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature; D014422:Turkeys / Q000821:virology*
chemical_list: D004396:Coloring Agents; D009282:Naphthalenesulfonates; D012367:RNA, Viral; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate