Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
J Virol Methods. 2010 Oct;169(1):8-12. doi: 10.1016/j.jviromet.2010.06.005. Epub 2010 Jun 23.
RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.
从环境样本中提取 RNA 通常会得到一种含有分子反应抑制剂的 RNA 制剂。商业 RNA 提取试剂盒通常允许提取的样品体积仅为 0.1-0.2ml。本研究设计了一种 RNA 提取缓冲液（RNAX 缓冲液），用于基于硅胶柱的方案从 4.0ml 中提取病毒 RNA。为了评估基于 RNAX 缓冲液的方案，我们使用甲型肝炎病毒 (HAV) 和柯萨奇病毒 B3 (CVB3) 来监测从环境样本中提取 RNA 的效率。为了评估通过 PEG 浓缩从河水和池塘水中制备的水样浓缩物中回收病毒 RNA 的情况，将两种水样病毒的连续十倍稀释液加入到水样浓缩物中，通过定量检测进行评估。通过逆转录定量实时 PCR (RT-qPCR) 定量回收 HAV 和 CVB3。提取的 RNA 与 RT-qPCR 兼容，使用 RNAX 缓冲液和开发的方案，检测到每反应 0.8PFU 的灵敏度。使用从接种水样浓缩物的 4.0ml 中提取的病毒 RNA 获得了这种灵敏度。本研究中开发的 RNAX 缓冲液可适用于检测水和食品中的其他病原体。
