Journal of Integrative Agriculture ›› 2015, Vol. 14 ›› Issue (8): 1637-1643.DOI: 10.1016/S2095-3119(15)61134-0

• 论文 • 上一篇    下一篇

A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms

 ZHANG Yong-qiang, LIU Hai-sheng, WU Xiao-dong, WANG Xiao-zhen, LI Jin-ming, ZHAO Yonggang, LÜ Yan, REN Wei-jie, GE Sheng-qiang, WANG Zhi-liang   

  1. 1、China Animal Health and Epidemiology Center, Qingdao 266032, P.R.China
    2、Beijing Dabeinong Technology Group Co., Ltd., Beijing 100080, P.R.China
  • 收稿日期:2014-10-18 出版日期:2015-08-05 发布日期:2015-08-06
  • 通讯作者: WANG Zhi-liang, E-mail: zlwang111@163.com
  • 作者简介:ZHANG Yong-qiang, E-mail: zhangyongqiang@cahec.cn;* These authors contributed equally to this study.
  • 基金资助:

    This research was supported by the China Agriculture Research System (CARS-37).

A novel real-time RT-PCR with TaqMan-MGB probes and its application in detecting BVDV infections in dairy farms

 ZHANG Yong-qiang, LIU Hai-sheng, WU Xiao-dong, WANG Xiao-zhen, LI Jin-ming, ZHAO Yonggang, LÜ Yan, REN Wei-jie, GE Sheng-qiang, WANG Zhi-liang   

  1. 1、China Animal Health and Epidemiology Center, Qingdao 266032, P.R.China
    2、Beijing Dabeinong Technology Group Co., Ltd., Beijing 100080, P.R.China
  • Received:2014-10-18 Online:2015-08-05 Published:2015-08-06
  • Contact: WANG Zhi-liang, E-mail: zlwang111@163.com
  • About author:ZHANG Yong-qiang, E-mail: zhangyongqiang@cahec.cn;* These authors contributed equally to this study
  • Supported by:

    This research was supported by the China Agriculture Research System (CARS-37).

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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 1.72×102 copies for BVDV 1 and 2.14×102 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 2 327 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.

关键词: bovine viral diarrhea virus (BVDV) , real-time RT-PCR , persistently infected (PI) animals , TaqMan-MGB , occurrence rate of PI cattle

Abstract: 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 1.72×102 copies for BVDV 1 and 2.14×102 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 2 327 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.

Key words: bovine viral diarrhea virus (BVDV) , real-time RT-PCR , persistently infected (PI) animals , TaqMan-MGB , occurrence rate of PI cattle