National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory, 4301 Rickenbacker Causeway, Miami, FL 33149, USA.
Water Res. 2013 Nov 15;47(18):6883-96. doi: 10.1016/j.watres.2013.02.059. Epub 2013 Jul 5.
Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions.
我们报告了一项多实验室（n=11）评估的结果，该评估针对的是最初开发用于检测沿海环境中海鸥粪便污染的 Catellicoccus marimammalium 的 16S rRNA 基因的四种不同 PCR 方法，包括常规终点 PCR 方法、SYBR(®)Green qPCR 方法和两种 TaqMan(®)qPCR 方法。测试了不同的数据归一化和分析技术。数据分析方法对检测限（LLOQ）、目标检测但不可量化（DNQ）和目标未检测（ND）的计算有显著影响。与各实验室在定义标准化之前提交的结果相比，包括定量下限（LLOQ）、目标检测但不可量化（DNQ）和目标未检测（ND）在内的指标的跨实验室标准化以及目标未检测（ND）在内的指标的跨实验室标准化显著提高了结果。用于数据归一化的度量单位也对测量的检测性能有显著影响。与肠球菌归一化相比，将数据归一化为 DNA 质量可提高定量方法的性能。本研究中测试的 MST 方法最初是为海鸥设计的，但在这项研究中也检测到了来自其他鸟类的粪便，特别是来自鸽子的粪便。测序结果表明，来自加利福尼亚的一些鸽子粪便中含有与在海鸥粪便中发现的 C. marimammalium 相似的序列。这些数据表明，C. marimammalium 在海鸥以外的宿主鸟类中的流行率、地理范围和生态学需要进一步研究。本研究代表了对这些方法进行多实验室评估的重要第一步，并强调需要进一步扩大和标准化其他评估，包括来自不同地理区域的环境相关目标浓度。
