Isaacs S T, Tessman J W, Metchette K C, Hearst J E, Cimino G D
HRI Research, Inc., Berkeley, CA 94710.
Nucleic Acids Res. 1991 Jan 11;19(1):109-16. doi: 10.1093/nar/19.1.109.
We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.
我们开发了一种有效的PCR后灭菌方法，并将该方法应用于HIV-1诊断检测。该方法基于异补骨脂素光化学原理，满足了实际灭菌过程中的灭活和杂交要求。该技术的关键特性是使用与DNA形成共价光化学加合物的异补骨脂素化合物。这些共价加合物可防止Taq聚合酶对先前扩增序列（扩增子）的后续延伸。异补骨脂素对PCR的抑制作用极小，可被长波长紫外线激活，能提供足够数量的共价加合物以实现有效灭菌，可修饰扩增序列使其保持单链状态，且对后续杂交影响很小。该灭菌方法可应用于封闭系统，适用于常用的检测形式。我们设计的光化学灭菌方案是消除与PCR相关的扩增子残留问题的一种有效且实用的方法。虽然此处所述工作仅限于HIV-1，但该技术的正确使用将消除与多种扩增子残留相关的担忧，尤其是在临床环境中。
