Marley Mylissa S D, Givens M Daniel, Galik Patricia K, Riddell Kay P, Stringfellow David A
Department of Pathobiology, College of Veterinary Medicine, 127 Sugg Laboratory, Auburn University, Auburn, AL 36849, USA.
Theriogenology. 2008 Jul 15;70(2):153-60. doi: 10.1016/j.theriogenology.2008.03.007. Epub 2008 May 2.
The objective of this study was to develop a duplex quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) type I and type II. Follicular fluid was collected from a BoHV-1 acutely infected heifer, a BVDV I persistently infected heifer, and from 10 ovaries recovered from an abattoir. Both the BoHV-1 and BVDV contaminated follicular fluid were diluted 1:5 to 1:10(7) using the pooled, abattoir-origin follicular fluid. Each dilution sample was analyzed using the duplex qPCR, virus isolation, reverse transcription-nested PCR (RT-nPCR), and BoHV-1 qPCR. The duplex qPCR was able to simultaneously detect BoHV-1 and BVDV I in the fluid diluted to 1:100 and 1:1000, respectively. These results corresponded with the reverse transcription-nested PCR and BoHV-1 qPCR. Therefore, the duplex qPCR might be used for quality assurance testing to identify these two viruses in cells, fluids and tissues collected from donor animals and used in reproductive technologies.
本研究的目的是开发一种双重定量聚合酶链反应（qPCR）检测方法，用于同时检测牛疱疹病毒1型（BoHV-1）和I型及II型牛病毒性腹泻病毒（BVDV）。从一头急性感染BoHV-1的小母牛、一头持续感染BVDV I的小母牛以及从屠宰场回收的10个卵巢中采集卵泡液。使用来自屠宰场的混合卵泡液将受BoHV-1和BVDV污染的卵泡液均稀释至1:5至1:10(7)。使用双重qPCR、病毒分离、逆转录巢式PCR（RT-nPCR）和BoHV-1 qPCR对每个稀释样本进行分析。双重qPCR能够分别在稀释至1:100和1:1000的液体中同时检测到BoHV-1和BVDV I。这些结果与逆转录巢式PCR和BoHV-1 qPCR的结果一致。因此，双重qPCR可用于质量保证检测，以鉴定从供体动物采集并用于生殖技术的细胞、液体和组织中的这两种病毒。
