Use of an exogenous plasmid standard and quantitative PCR to monitor spatial and temporal distribution of Enterococcus spp. in beach sands.
Use of an exogenous plasmid standard and quantitative PCR to monitor spatial and temporal distribution of Enterococcus spp. in beach sands.
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DOI：
10.4319/lom.2010.8.146
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发表时间：
2010-04
期刊：
影响因子：
--
通讯作者：
Gast RJ
中科院分区：
文献类型：
--
作者：
Halliday E;Griffith JF;Gast RJ
使用依赖培养的方法的研究表明，肠球菌是用于监测海洋水域对游泳者的肠道疾病潜在风险的粪便指标，可在海滩沙中大量存在，并可能导致水柱指标超标。建立了一种检测肠球菌属细菌的定量聚合酶链式反应(QPCR)方法，并将其应用于滩涂沙地中肠球菌数量的快速测定和时空分布的研究。该方法扩增了肠球菌特异的23S rDNA序列，并用于检测来自加利福尼亚州阿瓦隆湾的干湿海滩沙的亚样和复合样品。在qPCR反应中，DNA回收效率和抑制效率的差异是通过在提取前或提取后分别用已知数量的pGEM质粒添加对重复亚样来解释的。这项研究揭示了在湿沙和干沙中相似的环境抑制水平，以及DNA回收效率在潮湿海滩沙中显著较低，并且在不同年份之间存在差异。利用该方法产生的校正因子来估计肠球菌的丰度，我们发现湿的和干燥的海滩沙都有肠球菌。可能每天变化很大的种群，而且往往可能高于娱乐水域规定的同等健康标准。
Studies using culture dependent methods have indicated that enterococci, the fecal indicator used to monitor marine waters for the potential of enteric disease risk to swimmers, can be abundant in beach sands and may contribute to water column indicator exceedances. A quantitative PCR (qPCR) method for the Enterococcus genus was tested and applied to more rapidly determine the amount of enterococci in beach sands and study their distribution over space and time. The qPCR method amplified a 23S rDNA sequence specific to Enterococcus, and was used to examine subsamples and composite samples of wet and dry beach sand from Avalon Bay, CA. The differences in efficiency of DNA recovery and inhibition in qPCR reactions were accounted for by spiking pairs of duplicate subsamples with a known amount of pGEM plasmid before or after extraction, respectively. This study revealed levels of environmental inhibition that were similar in wet and dry sands, and efficiency of DNA recovery that was observably lower for wet beach sands and varied between years. Using the correction factors generated by this method to estimate the abundance of Enterococcus, we show that wet and dry beach sands both have Enterococcus spp. populations that can vary dramatically from day to day, and often are potentially higher than the equivalent health standards mandated for recreational waters.
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