Reynolds K A, Gerba C P, Abbaszadegan M, Pepper L L
Department of Soil, Water and Environmental Science, The University of Arizona, Tucson 85745, USA.
Can J Microbiol. 2001 Feb;47(2):153-7.
This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.
本研究应用细胞培养/聚合酶链反应综合方法（ICC/PCR）对致细胞病变和非致细胞病变病毒进行快速、特异性检测。本研究结果表明，单独使用直接逆转录聚合酶链反应（RT-PCR）或传统细胞培养方法分析环境样本时，可能会得出错误结果。本研究的目的是比较培养法、分子法和联合检测法，以找出在不同环境样本中检测病毒的最有效方法。使用ICC/PCR，在细胞培养中孵育仅5小时后，≥10个空斑形成单位（PFU）的肠道病毒接种原液在至少4/5个重复培养瓶中PCR呈阳性，孵育≥10小时后在所有培养瓶中均呈阳性。细胞培养孵育20小时后，通过PCR检测到1个PFU的接种量，而对于低于1个PFU的病毒浓度，25小时的孵育就足够了。同样，每瓶100个最可能数（MPN）的甲型肝炎病毒（HAV）接种物在细胞培养中产生不确定的细胞病变效应（CPE），但孵育48小时后通过ICC/PCR可清晰检测到。分别在孵育96至72小时后，通过ICC/PCR检测到较低水平的HAV，即1个MPN和10个MPN。与单独通过直接RT-PCR检测出3份阳性相比，来自污水、海水和地表饮用水源的11份环境样本浓缩物的细胞培养裂解物通过ICC/PCR检测肠道病毒呈阳性。ICC/PCR的结果最终与细胞培养结果一致，但孵育时间≤48小时，而与BGM和FRhK细胞孵育后出现CPE的时间长达3周。
