van Engelenburg F A, Maes R K, van Oirschot J T, Rijsewijk F A
Department of Virology, Central Veterinary Institute, Lelystad, The Netherlands.
J Clin Microbiol. 1993 Dec;31(12):3129-35. doi: 10.1128/jcm.31.12.3129-3135.1993.
We developed a polymerase chain reaction (PCR) assay to detect bovine herpesvirus type 1 (BHV-1) in bovine semen. Since bovine semen contains components that inhibit PCR amplification, a protocol was developed to purify BHV-1 DNA from bovine semen. To identify failures of PCR amplification, we used an internal control template that was coamplified by the same PCR primers. When separated fractions of BHV-1-contaminated semen were analyzed by the PCR, we found that more than 90% of the BHV-1 DNA was present in a pooled fraction consisting of seminal fluid, nonsperm cells, and virus adsorbed to spermatozoa. By using this fraction, three to five molecules of BHV-1 DNA in 50 microliters of bovine semen could be detected. A pilot study to compare this PCR assay with the routinely used virus isolation method showed that this PCR assay is 2- to 100-fold more sensitive. In addition, the results of the PCR assay are available in 1 day, whereas the virus isolation method takes 7 days. Therefore, the PCR assay may be a good alternative to the virus isolation method.
我们开发了一种聚合酶链反应（PCR）检测方法，用于检测牛精液中的牛疱疹病毒1型（BHV-1）。由于牛精液中含有抑制PCR扩增的成分，因此制定了一种从牛精液中纯化BHV-1 DNA的方案。为了识别PCR扩增失败的情况，我们使用了一个由相同PCR引物共同扩增的内部对照模板。当通过PCR分析BHV-1污染精液的分离部分时，我们发现超过90%的BHV-1 DNA存在于一个由精液、非精子细胞和吸附在精子上的病毒组成的混合部分中。使用该部分，在50微升牛精液中可以检测到三到五个BHV-1 DNA分子。一项将该PCR检测方法与常规使用的病毒分离方法进行比较的初步研究表明，该PCR检测方法的灵敏度高2至100倍。此外，PCR检测方法的结果在1天内即可获得，而病毒分离方法则需要7天。因此，PCR检测方法可能是病毒分离方法的一个很好的替代方法。
