Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye.
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.
Cardoso
Tereza C
TC
UNESP - São Paulo State University, Laboratory of Animal Virology, Campus de Araçatuba, São Paulo, Brazil. tcardoso@fmva.unesp.br
Ferrari
Heitor F
HF
Bregano
Lívia C
LC
Silva-Frade
Camila
C
Rosa
Ana Carolina G
AC
Andrade
Alexandre L
AL
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*
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