Saeki K, Mishima K, Horiuchi K, Hirota S, Nomura S, Kitamura Y, Aozasa K
Department of Pathology, Nara Medical University, Kashihara, Japan.
Diagn Mol Pathol. 1993 Jun;2(2):108-15.
A highly sensitive in situ hybridization procedure was established using digoxigenin-11-dUTP-labeled probes that were prepared by the polymerase chain reaction (PCR). By using 12 sets of primers, BamHI-W fragment of the Epstein-Barr Virus (EBV) was amplified with labeled substrate in individual PCRs. Then the 12 probes (average size, 120 base pairs) were mixed and hybridized with cultured and RNase-treated Namalwa cells, which contain two copies of EBV genomes per cell. The strength of the signals was much stronger as compared with random-primed probe. Our results indicate that the size-averaged PCR probes magnified the sensitivity for detecting low copy numbers of virus genomes by in situ hybridization and that this technique has the potential for investigating latent virus infection in other clinical situations.
利用聚合酶链反应（PCR）制备的地高辛-11-dUTP标记探针，建立了一种高度灵敏的原位杂交方法。通过使用12组引物，在单独的PCR反应中用标记底物扩增爱泼斯坦-巴尔病毒（EBV）的BamHI-W片段。然后将这12种探针（平均大小为120个碱基对）混合，并与经培养和核糖核酸酶处理的Namalwa细胞杂交，每个细胞含有两份EBV基因组。与随机引物探针相比，信号强度要强得多。我们的结果表明，大小平均的PCR探针通过原位杂交放大了检测低拷贝数病毒基因组的灵敏度，并且该技术有潜力用于研究其他临床情况下的潜伏病毒感染。
