DVGW-Technologiezentrum Wasser (TZW), Department Microbiology and Molecular Biology, Karlsruher Str. 84, Karlsruhe, Germany.
Chair of Analytical Chemistry and Institute of Hydrochemistry, Technical University of Munich, Marchioninistr. 17, 81377 Munich, Germany.
Water Res. 2016 Oct 15;103:141-148. doi: 10.1016/j.watres.2016.07.032. Epub 2016 Jul 15.
Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection.
水中病毒在风险评估方案中越来越受到重视。一般来说，病毒的培养方法检测比较耗时。相比之下，定量聚合酶链反应（qPCR）检测更加快速，因此更适合监测。目前，qPCR 缺乏区分传染性和非传染性病毒的能力，从而限制了其在消毒过程监测中的应用。本研究开发了一种通过长扩增子（LA）-qPCR 定量紫外线灭活的方法。噬菌体 PhiX174 被用作人类致病病毒的替代物。使用新的引物组开发了 qPCR 方案，扩增子长度为 108、250、456、568、955、1063、1544 和 1764 个核苷酸。基因拷贝数的对数减少随着扩增子长度的增加而增加。用嵌入染料 PMA 进行额外处理没有效果，表明噬菌体衣壳没有因低压紫外线照射而受损。对几乎完整基因组（约 5000 个核苷酸）的 qPCR 显示出与噬菌斑测定相似的结果。qPCR 中的对数减少与[特定扩增子长度 x UV 剂量]相关。归一化 DNA 效应常数可用于根据 qPCR 检测计算 PhiX174 的失活。
