Cuthbertson G, Grose C
Department of Pediatrics, University of Iowa College of Medicine, Iowa City 52242.
Mol Cell Probes. 1988 Sep;2(3):197-207. doi: 10.1016/0890-8508(88)90004-7.
We have developed and compared two DNA dot hybridization methodologies with similar probes (radioisotope-labelled and biotin-labelled) to detect varicella-zoster virus (VZV) DNA in three different cell cultures at varying times post-infection. Control cultures included uninfected monolayers of the same cells. Cellular DNA was isolated by a standard phenol extraction method, after which the DNA was quantified, serially diluted and blotted onto nitrocellulose or nylon membranes. The VZV DNA probe, which consisted of the large Hind III A fragment (27 of the total 125 kbp), was produced in two separate nick translation systems. The first contained 100 microCi [32P] and 0.4 microgram Hind III fragment A of the varicella genome, while the second probe employed a biotin-7-dATP analogue and 1.0 microgram of the Hind III fragment A. Direct visualization on the membrane or the exposed radiographic film showed a dot of varying intensity whenever viral genome was detected with either the biotin or the radioactive probe, respectively. With the [32P]-labelled probe, we detected VZV genomic sequences within 0.5 microgram total DNA at 12 h post-infection. This amount corresponded to approximately 5-10 pg of viral DNA. By comparison, hybridization with the biotin-labelled probe required 0.5-1.0 micrograms total DNA from infected cells. Similar tests on DNA extracted from uninfected cell samples were negative with both probes.
我们开发并比较了两种使用相似探针（放射性同位素标记和生物素标记）的DNA点杂交方法，以检测三种不同细胞培养物在感染后不同时间的水痘带状疱疹病毒（VZV）DNA。对照培养物包括相同细胞的未感染单层细胞。通过标准酚提取法分离细胞DNA，之后对DNA进行定量、连续稀释并点样到硝酸纤维素或尼龙膜上。VZV DNA探针由大的Hind III A片段（总共125 kbp中的27 kbp）组成，在两个独立的缺口平移系统中产生。第一个系统包含100微居里的[32P]和0.4微克水痘基因组的Hind III片段A，而第二个探针使用生物素-7-dATP类似物和1.0微克的Hind III片段A。当分别用生物素或放射性探针检测到病毒基因组时，在膜上或曝光的放射自显影片上直接观察到强度不同的点。使用[32P]标记的探针，我们在感染后12小时检测到0.5微克总DNA中的VZV基因组序列。这个量相当于约5-10皮克的病毒DNA。相比之下，用生物素标记的探针进行杂交需要来自感染细胞的0.5-1.0微克总DNA。对从未感染细胞样品中提取的DNA进行的类似测试，两种探针的结果均为阴性。
