扩增病毒DNA片段的微孔板杂交
Microplate hybridization of amplified viral DNA segment.
作者信息
Inouye S, Hondo R
机构信息
Department of Microbiology, University of Tokyo, Japan.
出版信息
J Clin Microbiol. 1990 Jun;28(6):1469-72. doi: 10.1128/jcm.28.6.1469-1472.1990.
Abstract
We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the amplified DNA segment with a length of more than 300 bases is adsorbed to microplate wells in the presence of 1.5 M NaCl or 0.5 M ammonium sulfate; the immobilized DNA is hybridized with a biotin-labeled DNA probe; then, the hybridization signal is detected by streptavidin-conjugated beta-galactosidase or peroxidase. This method has several advantages over the conventional dot blot hybridization method: (i) radioisotopes are not used, (ii) synthetic oligonucleotide for the probe is not needed, (iii) the time required for washing of the solid phase is greatly reduced, and (iv) the baking and prehybridization procedures are eliminated. By this method, we were able to detect viral genomes in vesicle specimens from patients infected with varicella-zoster virus.
摘要
我们开发了一种针对经聚合酶链反应扩增的DNA片段的简单杂交方法：热变性后，长度超过300个碱基的扩增DNA片段在1.5 M氯化钠或0.5 M硫酸铵存在的情况下吸附到微孔板孔中；固定化的DNA与生物素标记的DNA探针杂交；然后，通过链霉亲和素偶联的β-半乳糖苷酶或过氧化物酶检测杂交信号。该方法相对于传统的点杂交方法具有多个优点：（i）不使用放射性同位素，（ii）不需要合成寡核苷酸作为探针，（iii）固相洗涤所需时间大大减少，（iv）省去了烘烤和预杂交步骤。通过这种方法，我们能够在水痘-带状疱疹病毒感染患者的水疱标本中检测到病毒基因组。
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