期刊
JOURNAL OF VIROLOGICAL METHODS
卷 159, 期 1, 页码 64-68出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2009.03.001
关键词
Unclassified bovine enteric calicivirus; SYBR Green real-time RT-PCR; Quantitation; Rapid detection
资金
- National Veterinary Research and Quarantine Service (NVRQS) of the Ministry of Agriculture and Forestry
- Ministry of Commerce, Industry and Energy (MOCIE), Republic of Korea [RT105-01-01]
- Korean Ministry of Education and Human Resources Development
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 100 copies/mu l (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/mu l, 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. (C) 2009 Elsevier B.V. All rights reserved.
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