Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus

Abstract

Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n = 118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1×100 copies/μl (correlation coefficiency = 0.98). The detection limits of the RT-PCR and nested PCR were 1.1×105 and 1.1×102 copies/μl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.