Measurement of Enterococcus spp. levels with qPCR allows for same-day advisory notification of recreational water quality conditions, representing a major advance over traditional culture-based methods that require 18 or more hours to obtain results. In 2015, the United States Environmental Protection Agency released an Enterococcus qPCR protocol for recreational water quality testing. Over the past decade, there have been multiple advances in qPCR-based environmental testing, affording the opportunity to update the current methodology. A streamlined Enterococcus qPCR protocol is introduced that simplifies the mathematical model to estimate target sequence concentrations (TSC), reduces sample testing time by 20 min, incorporates a certified control material for standard curve generation, and introduces an inactivated E. faecalis whole cell DNA standard (WCDS) control material. A series of experiments were conducted 1) to compare results of the two Enterococcus qPCR protocols in analysis of marine, estuarine, and freshwater samples (n = 60), 2) to investigate alternative sources for sample processing control baseline measurements to identify and adjust results due to potential water sample matrix interference, and 3) to evaluate the performance, homogeneity, and stability of an inactivated E. faecalis cell preparation as a WCDS control material. Findings indicate a strong correlation between water sample mean log₁₀ TSC per reaction paired measurements (R² = 0.980) and 100% agreement in amplification and sample processing control tests. A Bayesian approach that accounts for uncertainty in qPCR measurements confirmed statistical equivalence for all water samples yielding paired measurements in the range of quantification, with 72.7% of samples exhibiting reduced error with the new streamlined protocol. Evaluation of three alternative sources to adjust for variation in Enterococcus qPCR measurements indicated no significant difference in water sample log₁₀ TSC per reaction results with varying concentrations of treated sewage influent. Systematic testing of an inactivated WCDS control material yielded statistically equivalent performance compared to viable E. faecalis cell preparations. Homogeneity and stability experiments indicated that Enterococcus qPCR measurements of inactivated WCDS are reproducible across multiple preparations and that the material is stable at -20°C for at least 38 weeks. Together, experiments demonstrate that the streamlined protocol and alternative practices should make Enterococcus qPCR faster, easier to implement, safer, and more reproducible.

中文翻译：

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一种用于表征肠球菌属的简化 qPCR 方法。环境地表水样品中的含量

使用 qPCR 测量肠球菌属水平，可在当天通知娱乐用水质量状况，这比需要 18 小时或更长时间才能获得结果的传统基于培养的方法取得了重大进步。2015 年，美国环境保护署发布了用于娱乐用水质量检测的肠球菌 qPCR 方案。在过去十年中，基于 qPCR 的环境检测取得了多项进展，为更新当前方法提供了机会。引入了一种简化的肠球菌 qPCR 方案，该方案简化了估计靶序列浓度 （TSC） 的数学模型，将样品检测时间缩短了 20 分钟，加入了用于标准曲线生成的认证对照材料，并引入了灭活的粪肠球菌全细胞 DNA 标准 （WCDS） 对照材料。进行了一系列实验：1） 比较两种肠球菌 qPCR 方案在海洋、河口和淡水样品分析中的结果 （n = 60），2） 研究样品处理对照基线测量的替代来源，以识别和调整由于潜在水样基质干扰引起的结果，以及 3） 评估性能、均一性、 以及灭活的粪肠球菌细胞制剂作为 WCDS 对照材料的稳定性。研究结果表明，水样平均对数 ₁₀ TSC 每个反应配对测量值 （R² = 0.980） 与扩增和样品处理对照测试中的 100% 一致性之间存在很强的相关性。 考虑 qPCR 测量不确定性的贝叶斯方法证实了所有水样品的统计等效性，在定量范围内产生配对测量值，72.7% 的样品在新的简化方案中表现出减少的误差。对三种替代来源的评估以调整肠球菌 qPCR 测量的变化，表明在不同浓度的处理污水进水下，每个反应结果的水样对数 ₁₀ TSC 没有显著差异。与活的粪肠球菌细胞制剂相比，灭活 WCDS 对照材料的系统测试产生了统计学上等效的性能。均一性和稳定性实验表明，灭活 WCDS 的肠球菌 qPCR 测量在多种制备中是可重复的，并且该材料在 -20°C 下可稳定保存至少 38 周。总之，实验表明，简化的方案和替代实践应该使肠球菌 qPCR 更快、更容易实施、更安全且更具可重复性。