Hondo R, Ito S, Inouye S
Department of Microbiology, the Institute of Public Health, Tokyo.
Jpn J Med Sci Biol. 1995 Oct-Dec;48(5-6):249-55. doi: 10.7883/yoken1952.48.249.
We devised a simple procedure for titration of varicella-zoster virus (VZV) DNA in throat swabs from varicella patients. DNA which was extracted from throat swabs, together with known copy numbers of a cloned VZV DNA fragment, were 10-fold serially diluted and used as template in PCR. The PCR products, after heat denaturation, again serially diluted in 1.5 M NaCl and adsorbed to microplate wells. Then, biotin-labeled DNA probes were hybridized with the immobilized DNA. The hybridization signal was produced by streptavidin-conjugated beta-galactosidase and a fluorogenic enzyme substrate. By comparing the titration curves of a clinical specimen with those of the cloned fragment, of which detection limit was about 10 copies, we estimated the copy numbers of VZV DNA in the specimen. With this technique, we evaluated the degree of potential contagiousness of the patient along the course of infection: we found that varicella patients possessed highest quantity of VZV DNA in the throat on the first day of illness.
我们设计了一种简单的方法，用于滴定水痘患者咽拭子中的水痘带状疱疹病毒（VZV）DNA。从咽拭子中提取的DNA与已知拷贝数的克隆VZV DNA片段一起进行10倍系列稀释，并用作PCR模板。PCR产物经热变性后，再次在1.5M NaCl中进行系列稀释，并吸附到微孔板孔中。然后，生物素标记的DNA探针与固定化的DNA杂交。杂交信号由链霉亲和素偶联的β-半乳糖苷酶和荧光酶底物产生。通过比较临床标本与克隆片段的滴定曲线（克隆片段的检测限约为10个拷贝），我们估计了标本中VZV DNA的拷贝数。通过这项技术，我们评估了患者在感染过程中的潜在传染性程度：我们发现水痘患者在发病第一天喉咙中的VZV DNA数量最多。
