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首页> 外文期刊>Applied Microbiology >Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples
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Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

机译：靶向肠球菌16s rRNA基因的定量PCR分析方法的发展。及其在环境样品中肠球菌种类鉴定中的应用

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The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium , although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.
机译：主要通过使用基于文化的技术来确定环境肠球菌的检测，这些技术可能会排除某些肠球菌以及不可培养的肠球菌。为了解决这个问题，通过挑战粪便和水样，对照目前可用的属特异性测定法（Entero1）来检查肠球菌的相对丰度。为了确定肠球菌的多样性，开发了基于16S rRNA基因的组特异性定量PCR（qPCR）分析方法，并针对八种最常见的环境肠球菌进行了评估。分析了439种推定环境肠球菌菌株的部分16S rRNA基因序列，以进一步研究肠球菌的多样性并确认组特异性测定的特异性。特定于组的qPCR分析显示了针对目标物种的相对较高的扩增率（> 98％），尽管一些分析与非目标物种进行了交叉扩增（1.3％至6.5％）。组特异性测定的结果还表明，大多数粪便样品中同时存在不同的肠球菌。在水和粪便样品中，肠球菌含量最高的是粪肠球菌和粪便肠球菌，尽管我们鉴定出的水分离株比其他任何种类的肠球菌都多。除粪便外，大多数粪便样品中Entero1标记的阳性率与通过组特异性测定法确定的阳性信号总数相吻合。另一方面，在所有测试的水样品中，组特异性测定信号的数量均较少，表明这些样品中还存在其他肠球菌。虽然结果强调了属和组特异性测定法在检测环境水样品中主要肠球菌群的价值，但仍需要进一步的研究以确定水中发现的所有肠球菌物种的多样性，分布和相对丰度。

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