Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus.
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 x 10(0)copies/microl (correlation coefficiency=0.98). The detection limits of the RT-PCR and nested PCR were 1.1 x 10(5) and 1.1 x 10(2)copies/microl, 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.
Park
Sang-Ik
SI
Bio-therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South Korea.
Park
Da-Hae
DH
Saif
Linda J
LJ
Jeong
Young-Ju
YJ
Shin
Dong-Jun
DJ
Chun
Young-Hyun
YH
Park
Su-Jin
SJ
Kim
Hyun-Jeong
HJ
Hosmillo
Myra
M
Kwon
Hyung-Jun
HJ
Kang
Mun-Il
MI
Cho
Kyoung-Oh
KO
D000818:Animals; D052160:Benzothiazoles; D002139:Caliciviridae / Q000235:genetics / Q000302:isolation & purification*; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000821:virology; D003959:Diamines; D005243:Feces / Q000821:virology; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I