Naif H M, Daniel R C, Cougle W G, Lavin M F
Queensland Cancer Fund Research Unit, Bancroft Centre, Herston, Brisban, Australia.
J Clin Microbiol. 1992 Mar;30(3):675-9. doi: 10.1128/jcm.30.3.675-679.1992.
Bovine leukemia virus is the causative agent of bovine leukosis and has been described in many countries throughout the world. We describe here a sensitive and readily applicable assay for the detection of bovine leukemia proviral DNA. Detection relies on initial amplification of proviral DNA by using polymerase chain reaction (PCR) followed by an enzyme-linked assay (PCR-ELA). Amplification is carried out by using one biotinylated primer and a second primer containing the GCN4 protein binding site. DNA is detected by a colorimetric assay after it is coupled to GCN4-coated plates and subsequently incubated with horseradish-streptavidin peroxidase and the appropriate substrate to produce a chromogenic reaction. It was possible to detect proviral DNA for all of eight bovine leukemia virus-infected calves by 2 weeks postinfection. Use of the more conventional agar gel immunodiffusion assay failed to reveal the presence of the virus in any of the animals up to 4 weeks postinfection. The PCR-ELA detected as little as 0.1 to 0.2 ng of amplified DNA per well, which compares very favorably with ethidium bromide staining of gels, by which 1 to 2 ng per lane was detected. This method lends itself to mass screening, is carried out in a similar way to an enzyme-linked immunosorbent assay, and does not require gel electrophoresis or the use of radioactive gene probes.
牛白血病病毒是牛白血病的病原体，已在世界许多国家被发现。我们在此描述一种灵敏且易于应用的检测牛白血病前病毒DNA的方法。检测依赖于首先通过聚合酶链反应（PCR）扩增前病毒DNA，随后进行酶联检测（PCR - ELA）。扩增通过使用一条生物素化引物和另一条含有GCN4蛋白结合位点的引物来进行。DNA在与包被有GCN4的平板偶联后，通过比色法进行检测，随后与辣根 - 链霉抗生物素蛋白过氧化物酶及合适的底物一起孵育以产生显色反应。在感染后2周时能够检测到所有8头感染牛白血病病毒的犊牛的前病毒DNA。使用更传统的琼脂凝胶免疫扩散试验，在感染后长达4周的时间内，未能在任何动物中检测到病毒的存在。PCR - ELA每孔能够检测低至0.1至0.2 ng的扩增DNA，这与凝胶的溴化乙锭染色相比非常有利，后者每条泳道可检测到1至2 ng。该方法适用于大规模筛查，其操作方式与酶联免疫吸附测定类似，并且不需要凝胶电泳或使用放射性基因探针。
