Gard J A, Givens M D, Riddell K P, Galik P K, Zhang Y, Stringfellow D A, Marley M S D
Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, AL 36849, USA.
Theriogenology. 2007 Jun;67(9):1415-23. doi: 10.1016/j.theriogenology.2007.01.014. Epub 2007 Apr 8.
The objectives of this study were to develop techniques to detect BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using either virus isolation (VI) or real time quantitative polymerase chain reaction (RT-QPCR) assays. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed to SD-1, a high affinity strain of BVDV, for 2 h and then processed according to the International Embryo Transfer Society (IETS) guidelines prior to testing. Groups of five or two in vivo-derived embryos, and single in vivo-derived embryos, were VI positive for BVDV 100, 50, and 33% of the time, and were RT-QPCR positive 100, 75, and 42% of the time, respectively. The virus was detected by the VI technique in all of the groups of five or two in vitro-produced embryos and in all of the single in vitro-produced embryos, and it was detected in 100, 80, and 50%, using RT-QPCR. Techniques for RT-QPCR were sufficiently sensitive to detect 10 copies of viral RNA in a sample and to detect BVDV associated with single embryos. Application of this new technology, RT-QPCR, will facilitate additional studies to further assess the risk of transmission of BVDV through embryo transfer.
本研究的目的是开发技术，使用病毒分离（VI）或实时定量聚合酶链反应（RT-QPCR）检测法，来检测存在于少量培养基中的单个或小群牛胚胎相关的牛病毒性腹泻病毒（BVDV）。将受精后7天的体内来源和体外生产的牛胚胎暴露于高亲和力BVDV毒株SD-1 2小时，然后在检测前按照国际胚胎移植协会（IETS）指南进行处理。五组或两组体内来源的胚胎以及单个体内来源的胚胎，采用VI检测时，BVDV呈阳性的时间分别为100%、50%和33%，采用RT-QPCR检测时，呈阳性的时间分别为100%、75%和42%。采用VI技术在所有五组或两组体外生产的胚胎以及所有单个体外生产的胚胎中均检测到病毒，采用RT-QPCR检测时，检测到病毒的比例分别为100%、80%和50%。RT-QPCR技术灵敏度足够高，能够检测样品中10个病毒RNA拷贝，并能检测与单个胚胎相关的BVDV。这项新技术RT-QPCR的应用将有助于开展更多研究，以进一步评估BVDV通过胚胎移植传播的风险。
