科研之友
微信
新浪微博
Facebook
分享链接
摘要
A real-time RT-PCR assay using TaqMan-MGB probes was developed to detect and type the bovine viral diarrhea virus (BVDV) in cattle. Universal primers and TaqMan-MGB probes were designed from the 5-untranslated region of known pestiviral sequences. Prior to optimizing the assay, cRNAs were transcribed in vitro from the BVDV 1 and BVDV 2 RTPCR products to make standard curves. The detection limit of the assay was 1.72x10(2) copies for BVDV 1 and 2.14x10(2) copies for BVDV 2. The specificity of the assay evaluated on several BVDV strains including bovine herpesvirus 1 (BHV 1), foot and mouth disease virus (FMDV) and several classical swine fever virus (CSFV) strains showed specific detection of the positive virus over 40 cycles. The assay was highly reproducible with the coefficient of variance ranging from 1.04 to 1.33% for BVDV 1 and from 0.83 to 1.48% for BVDV 2, respectively. Using this method, we tested a total of 2327 cattle from three dairy farms for the presence of BVDV persistently infected (PI) animals. In this assay, each RT-PCR template contained a mixture of ten samples from different animals. The occurrence rate of PI cattle in three farms ranging from 0.9 to 2.54% could represent partly the PI rates in cattle farm in China. In conclusion, using our real-time PCR assay, we could effectively detect and type BVDV and identify PI cattle in a rapid and cost-effective manner.
- 出版日期2015
- 单位中国动物卫生与流行病学中心
