Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA.
J Virol Methods. 2012 Apr;181(1):43-50. doi: 10.1016/j.jviromet.2012.01.009. Epub 2012 Feb 3.
Quantitative real-time PCR (qPCR) is used commonly to detect adenovirus (Ads) and norovirus (Nvs) in recreational waters. However, qPCR detection may be limited by interference from inhibitory substances found in recreational waters. In previous work, viruses in Avalon and Doheny Beach water samples were concentrated by electropositive cartridge filtration and PEG precipitation, and high inhibition was found in the samples when using qPCR for detection of Ads and Nvs. Therefore, different approaches were evaluated for removal or blocking of inhibitory compounds that affect qPCR. Avalon and Doheny concentrates were spiked with known amounts of Ads 41 and Nvs GII, and spiked deionized water was used as a positive control. Modifications included gel chromatography with columns of Sephadex G-200/Chelex 100, different sample volumes for nucleic acid extraction, organic solvent extraction, and nucleic acid precipitation. The efficiency of each treatment varied according to sampling location and virus type. The best option for improved Nvs detection by reverse transcription-qPCR was to reduce the sample volume for nucleic acid purification. The best option for improving Ads detection in both beach samples was Sephadex/Chelex spin column chromatography. Chloroform extraction only improved virus detection in Doheny Beach samples but not in Avalon Beach samples. Observed differences in effective treatments between viruses may be related to the different PCR targets, amplification conditions, and enzymes used in each assay, and differences between beaches may be related to differences in PCR inhibitory environmental compounds at each location. The results suggest that methods for detecting viruses from marine beaches, including treatments for the removal of PCR inhibitory compounds, should be optimized for each sampling site and probably for each virus of interest.
定量实时聚合酶链反应 (qPCR) 常用于检测娱乐水中的腺病毒 (Ads) 和诺如病毒 (Nvs)。然而，qPCR 检测可能会受到娱乐水中抑制物质的干扰。在之前的工作中，通过正电荷筒式过滤和聚乙二醇沉淀浓缩 Avalon 和 Doheny 海滩水样中的病毒，当使用 qPCR 检测 Ads 和 Nvs 时，发现这些水样中的抑制作用很高。因此，评估了不同的方法来去除或阻断影响 qPCR 的抑制性化合物。将已知量的 Ads 41 和 Nvs GII 加入 Avalon 和 Doheny 浓缩物中，并将加标去离子水作为阳性对照。修改包括使用 Sephadex G-200/Chelex 100 柱的凝胶色谱法、核酸提取的不同样品体积、有机溶剂提取和核酸沉淀。根据采样地点和病毒类型的不同，每种处理方法的效率也有所不同。通过逆转录-qPCR 提高 Nvs 检测效率的最佳选择是减少核酸纯化的样品体积。提高两个海滩样本中 Ads 检测的最佳选择是 Sephadex/Chelex 螺旋柱色谱法。氯仿提取仅提高了 Doheny 海滩样本中病毒的检测，但并未提高 Avalon 海滩样本中病毒的检测。在不同病毒中观察到的有效处理方法之间的差异可能与每个检测中使用的不同 PCR 靶标、扩增条件和酶有关，海滩之间的差异可能与每个地点的 PCR 抑制性环境化合物的差异有关。结果表明，应针对每个采样点（可能还有每个感兴趣的病毒）优化用于检测海洋海滩病毒的方法，包括去除 PCR 抑制性化合物的处理方法。
