Real-time RT-PCR of feline calicivirus and optimization for detection of virus in feline urine.

摘要

Investigating the role of feline calicivirus (FCV) in idiopathic cystitis may be facilitated by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay optimized for detection of FCV urinary tract infections. Two FCV RT-PCR assays were developed; a p5.6 gene-based qualitative assay and a p30 gene-based quantitative real-time SYBRRTM Green I assay. The p5.6 gene assay was highly sensitive and specific, but was not efficiently adapted to real-time RT-PCR. The real-time p30 gene assay was sensitive, specific, and linear over a wide range of template concentrations, and had a reaction efficiency of 95%. The p30 gene assay detected all 51 North American FCV field isolates tested. To optimize detection of FCV in urine by RT-PCR, viral RNA was prepared from urine by dilution and thermal inactivation, polyethylene glycol precipitation, isolation with oligo(dT)25-coated magnetic beads, or extraction with two silica gel-based columns. The FCV real-time p30 gene assay performed significantly better when using RNA isolated from feline urine with either of the silica gel-based columns.