监测饮用水中机会病原体的水平对于计划干预措施和了解允许它们增殖的生态位非常重要。定量PCR是一种已建立的替代培养方法,可以提供更快,更高的吞吐量,和更精确的水样中的细菌计数。然而,基于PCR的方法仍未常规用于军团菌监测,和技术,比如DNA提取,不同的实验室。这里,我们量化了DNA提取方法对下游PCR定量和群落测序的影响.通过社区科学运动,我们收集了50个水样和相应的淋浴软管,并将两种常用的DNA提取方法与相同的生物膜和水相样品进行了比较。两种方法显示出明显不同的提取效果,这反映在提取的DNA数量和两个基质中计数的军团菌浓度上。值得注意的是,一种方法导致几乎所有样品的更高计数约一个数量级,并在另一种方法未检测到的21个样品中检测到军团菌。16SrRNA扩增子测序显示,个体分类群的相对丰度,包括军团菌的序列变异,根据所采用的提取方法,差异很大。鉴于这些发现的含义,我们主张改进饮用水中用于检测和量化军团菌的DNA提取方法的性能记录,并表征相关的微生物群落。重要的是,监测水传播的机会性病原体军团菌的存在对于评估感染风险和计划补救措施非常重要。虽然监测传统上是通过种植进行的,对快速和高通量的基于分子的军团菌检测方法的需求不断增加。本文提供了有关DNA提取如何影响下游分子分析的有价值的见解,例如通过液滴数字PCR定量军团菌和通过测序分析表征天然微生物群落。我们分析了风险评估的结果,立法,和生态视角,显示初始DNA处理是如何在转向基于分子的常规监测时考虑的重要步骤,并讨论一致和详细报告所用方法的核心作用。
Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community
sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon
sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through
sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.