Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters
Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters
复制标题
DOI：
10.1128/aem.00651-10
复制
发表时间：
2010-11-01
影响因子：
4.4
通讯作者：
Weisberg, Stephen B.
中科院分区：
文献类型：
--
作者：
Noble, Rachel T.;Blackwood, A. Denene;Weisberg, Stephen B.
目前使用基于培养的方法监测娱乐用水的质量，监测结果需要18至96小时。定量聚合酶链式反应(QPCR)方法可以在不到2 h的时间内完成，但其结果与常规方法不同。我们提出了两项研究，其中的样本是同时处理肠球菌属。用两种基于培养的方法(EPA方法1600和Enterolt/Colilert-18)和QPCR法检测大肠埃希菌。专有的QPCR检测针对23S rRNA(肠球菌属)和uidA(大肠杆菌)基因，并使用含有所有试剂的冻干珠进行检测。在第一项研究中，QPCR方法的开发人员处理了54个盲样样本，这些样本接种了污水或纯培养物，或者是周围海滩的样本。第二项研究涉及163个由水质人员处理的样本。在第一次研究中，肠球菌的定量聚合酶链式反应结果与EPA 1600的相关性(r(2))为0.69，低于两种培养方法之间的相关性(r(2)，0.87)。在第二项研究中，两者的相关性是相似的。当对空白样本进行基于QPCR的检测时，这两项研究都没有出现假阳性。在两项研究中，通过变异系数测量的重复性水平与肠球菌定量聚合酶链式反应结果和基于培养的方法相似，但在第一项研究中，大肠杆菌定量聚合酶链式反应结果的重复性较高。关于加利福尼亚州发布海滩管理决定的集中程度，肠球菌QPCR法和EPA方法1600的结果之间的一致性为88%，而EPA方法1600和Enterolt方法的一致性为94%。大肠杆菌QPCR与Colilert-18的海滩管理决策符合率为94%。显示出不一致的样本表明对QPCR值估计偏低。
Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r(2)) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r(2), 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.