Wiedmann M, Brandon R, Wagner P, Dubovi E J, Batt C A
Department of Food Science Cornell University Ithaca, NY 14853.
J Virol Methods. 1993 Sep;44(1):129-39. doi: 10.1016/0166-0934(93)90015-j.
A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rapid and sensitive detection of the PCR products was achieved using a nonisotopic reverse dot-blot format with a visible color readout. Cross-reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant from a BHV-1 cell culture was approximately 4.5 TCID50 (50% tissue culture infectious dose). A procedure using the chelating resin Chelex 100 was used to prepare viral DNA from artificially inoculated samples of extended and raw semen for use in the PCR assay. In combination with nested PCR and reverse dot blot, this method allowed the detection of 5 x 10(3) TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in approximately 8 h. This assay has therefore the potential of replacing the currently available yet time consuming and costly detection methods for BHV-1 in bovine semen.
已开发出一种针对糖蛋白IV基因部分区域的巢式PCR检测方法，用于检测牛疱疹病毒1型（BHV-1）。使用具有可见颜色读数的非同位素反向斑点杂交形式实现了对PCR产物的快速灵敏检测。该PCR检测方法与密切相关的BHV-3未观察到交叉反应。在BHV-1细胞培养物的上清液上进行测试时，该检测方法的灵敏度约为4.5个TCID50（50%组织培养感染剂量）。使用螯合树脂Chelex 100的程序从人工接种的精液延长液和原精液样本中制备病毒DNA，用于PCR检测。结合巢式PCR和反向斑点杂交，该方法能够检测每0.5毫升精液中5×10(3)个TCID，这与康奈尔精液检测中的检测结果相当。整个过程大约可在8小时内完成。因此，该检测方法有可能取代目前用于检测牛精液中BHV-1的耗时且昂贵的现有检测方法。
