California Department of Public Health, Drinking Water and Radiation Laboratory Branch, 850 Marina Bay Pkwy G164, Richmond, CA 94804, United States of America.
San Diego County Department of Environmental Health, 5500 Overland Ave #170, San Diego, CA 92123, United States of America.
J Microbiol Methods. 2021 May;184:106206. doi: 10.1016/j.mimet.2021.106206. Epub 2021 Mar 23.
Droplet digital polymerase chain reaction (ddPCR) was evaluated for the detection of fecal indicator bacteria (FIB), Enterococcus spp., in San Diego County beach water samples collected under diverse conditions, from multiple pollution sources, as part of regulatory monitoring activities over 20 months. Two US EPA-approved methods, qPCR (EPA 1609.1) and Enterolert (SM9230D), were used as reference comparator methods. A total of 361 samples were assayed by both ddPCR and qPCR and yielded an acceptable Index of Agreement (IA) of 0.89, based on EPA Site-Specific analysis guidelines. A Pearson's correlation coefficient of r = 0.87 (p < 0.001), further indicated a strong relationship between the methods results. From the 361 samples, 185 split samples with ddPCR and Enterolert values within the limits of quantification, were used as a 'training' data set to derive an intrinsic copy number equation (ICE) for scaling ddPCR gene copy number to Enterolert most probable number (MPN). Of the 1993 samples that comprised the complete 'test' data set assayed by ddPCR and Enterolert, 1086 generated results that fell within the limits of quantification for Enterolert and yielded an overall IA of 0.64. Re-analysis using median as a measure of central tendency to account for significant skewing of Enterolert data yielded an IA of 0.72. Beach grouping-specific IA values ranged from 0.63 to 0.93. Pearson's correlation coefficient, r, ranged from 0.13 to 0.94 within beach groupings and generated a combined value of 0.60 for all groupings. Using the ICE, a ddPCR advisory threshold of 1413 DNA copy number/100 mL was empirically determined to be the equivalent to the California Enterolert beach action threshold of 104 MPN/100 mL, based on comparison with all 1993 paired ddPCR and Enterolert results. Using the 1413 DNA copy number/100 mL as a beach action threshold for ddPCR resulted in a 90.4% agreement with Enterolert (6.0% false negative and 3.7% false positive). Together these findings support the conclusion that ddPCR readouts align closely with Enterolert MPN for identifying FIB exceedance levels of Enterococcus spp. in coastal waters of San Diego, CA.
液滴数字聚合酶链反应（ddPCR）用于检测圣迭戈县海滩水样中的粪便指示菌（FIB）和肠球菌属，这些水样是在 20 多个月的监管监测活动中，从多个污染源在各种条件下采集的。两种美国环保署（EPA）批准的方法，qPCR（EPA 1609.1）和 Enterolert（SM9230D），被用作参考比较方法。共有 361 个样本通过 ddPCR 和 qPCR 进行了检测，根据 EPA 特定地点分析指南，得到了可接受的一致性指数（IA）为 0.89。Pearson 相关系数 r = 0.87（p < 0.001）进一步表明，这两种方法的结果具有很强的相关性。在 361 个样本中，185 个具有 ddPCR 和 Enterolert 定量限内值的分割样本被用作“训练”数据集，以推导出将 ddPCR 基因拷贝数转换为 Enterolert 最可能数（MPN）的内在拷贝数方程（ICE）。在由 ddPCR 和 Enterolert 检测的 1993 个样本的完整“测试”数据集中，有 1086 个结果落在 Enterolert 的定量限内，总体 IA 为 0.64。使用中位数作为衡量中心趋势的指标重新分析，以解释 Enterolert 数据的显著偏斜，得到的 IA 为 0.72。海滩分组特定的 IA 值范围为 0.63 至 0.93。Pearson 相关系数 r 在海滩分组内范围从 0.13 到 0.94，在所有分组中产生了 0.60 的综合值。使用 ICE，根据与所有 1993 对 ddPCR 和 Enterolert 结果的比较，确定 ddPCR 咨询阈值为 1413 DNA 拷贝数/100 mL，相当于加利福尼亚州 Enterolert 海滩行动阈值为 104 MPN/100 mL。使用 1413 DNA 拷贝数/100 mL 作为 ddPCR 的海滩行动阈值，与 Enterolert 相比，其结果为 90.4%（6.0%假阴性和 3.7%假阳性）。这些发现共同表明，ddPCR 的检测结果与 Enterolert 的 MPN 非常吻合，可用于识别加利福尼亚州圣迭戈沿海地区的肠球菌属 FIB 超过水平。
