Jothikumar N, Khanna P, Paulmurugan R, Kamatchiammal S, Padmanabhan P
National Environmental Engineering Research Institute (NEERI), Madras Zonal Laboratory, India.
J Virol Methods. 1995 Nov;55(3):401-15. doi: 10.1016/0166-0934(95)00089-9.
A simultaneous concentration of enteroviruses, hepatitis E virus, and rotavirus from drinking water samples through a filtration column filled with granular activated carbon (GAC) was achieved. Urea-arginine phosphate buffer (UAPB) as an eluent at pH 9.0 was used for effective desorption and elution of viruses from GAC. Further concentration of viruses with magnesium chloride enabled nucleic acid extraction, cDNA synthesis, amplification with a specific set of primers for enterovirus, hepatitis E virus and rotavirus. Polymerase chain reaction (PCR) products were then confirmed by Southern transfer and hybridization with the relevant probes. The efficacy of the protocol was established with 100 1 of water samples seeded with poliovirus-1, providing 74% recovery in granular activated carbon based UAPB-RT-PCR. The GAC-based method for concentration of viruses from water samples was preferred, despite its somewhat lower efficacy compared to 80% in membrane filter based UAPB-RT-PCR protocol, due to the specific requirements of short-time and savings in cost of analyses. The protocol was used for the detection of waterborne viruses from 24 drinking water sources in urban areas of New Delhi. Direct isolation of viruses from water samples revealed that the 4 samples were positive for enteroviruses, two for hepatitis E virus, and 10 samples for rotavirus. One sample was positive for both hepatitis E virus and rotavirus, and another for all the 3 types of viruses.
通过填充颗粒活性炭（GAC）的过滤柱，实现了从饮用水样本中同时浓缩肠道病毒、戊型肝炎病毒和轮状病毒。使用pH 9.0的磷酸尿素-精氨酸缓冲液（UAPB）作为洗脱液，可有效从GAC中解吸和洗脱病毒。用氯化镁进一步浓缩病毒后进行核酸提取、cDNA合成，并使用针对肠道病毒、戊型肝炎病毒和轮状病毒的特定引物组进行扩增。然后通过Southern转移并用相关探针杂交来确认聚合酶链反应（PCR）产物。用接种了脊髓灰质炎病毒-1的100升水样建立了该方案的有效性，在基于颗粒活性炭的UAPB-RT-PCR中回收率为74%。尽管与基于膜滤器的UAPB-RT-PCR方案中80%的回收率相比，基于GAC的从水样中浓缩病毒的方法效率略低，但由于分析时间短和成本节省的特定要求，该方法更受青睐。该方案用于检测新德里市区24个饮用水源中的水传播病毒。从水样中直接分离病毒显示，4个样本肠道病毒呈阳性，2个样本戊型肝炎病毒呈阳性，10个样本轮状病毒呈阳性。1个样本戊型肝炎病毒和轮状病毒均呈阳性，另1个样本3种病毒均呈阳性。
