Reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus

J Virol Methods. 2017 May:243:105-108. doi: 10.1016/j.jviromet.2017.01.009. Epub 2017 Jan 18.

Abstract

The Feline coronavirus (FCoV) is the etiological agent of feline infectious peritonitis (FIP), a lethal disease of felids. The role of molecular methods is controversial for the diagnosis of FIP, while essential for the identification of the shedders. Thus, a fast and inexpensive method for the detection of FCoV could be beneficial, especially in multicat environments. A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was developed. RNA extraction and RT-nPCR for FCoV were performed on thirty-two samples (11 faeces, 9 blood, 8 effusions, and 4 lymph nodes) collected from 27 cats. Six RT-LAMP primers were designed from the same conserved region of RT-nPCR, and the assay was run at 63°C for one hour. Results were evaluated through both agarose gel run and hydroxynapthol blue (HNB) dye and then compared with RT-nPCR results for the assessment of sensitivity and specificity. The overall specificity was 100%, but the sensitivity was 50% and 54.5% for agarose gel and HNB respectively. Therefore, RT-LAMP seems optimal to confirm the presence of the virus, but not applicable to exclude it.

Keywords: Feline infectious peritonitis; Hydroxynaphtol blue; Isothermal gene amplification.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Cats
  • Coronavirus, Feline / genetics
  • Coronavirus, Feline / isolation & purification*
  • DNA Primers / genetics
  • Feline Infectious Peritonitis / diagnosis*
  • Feline Infectious Peritonitis / virology
  • Nucleic Acid Amplification Techniques / methods*
  • RNA, Viral / analysis*
  • RNA, Viral / genetics
  • Reverse Transcription*
  • Sensitivity and Specificity
  • Temperature

Substances

  • DNA Primers
  • RNA, Viral