Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes
Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes
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DOI：
10.1111/j.1472-765x.2010.03001.x
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发表时间：
2011-03-01
影响因子：
2.4
通讯作者：
Haugland, R. A.
中科院分区：
文献类型：
--
作者：
Chern, E. C.;Siefring, S.;Haugland, R. A.
目的：比较特异性和灵敏度的定量PCR（qPCR）检测靶向单拷贝和多拷贝基因区域的Escherichiacoli.Methods和结果：以前报道的检测靶向uidA基因（uidA 405）被用作比较的基础上的分类特异性和灵敏度的qPCR检测靶向rodA基因（rodA 984）和两个区域的多拷贝23 S核糖体RNA基因（EC23 S和EC23 S857）。对28株代表E.结果表明，uidA 405和rodA 984对大肠杆菌和21种相关非靶种的特异性均为100%。而EC23 S检测仅为29%特异性。由于检测到E. fergusonii。用uidA 405、rodA 984、EC23 S和EC23 S857检测175株疑似大肠杆菌，敏感性分别为85%、85%、100%和86%。从淡水、海水和废水样本中分离的大肠杆菌。在对32份淡水、海水和废水样品的DNA提取物进行分析时，rodA 984、EC23 S和EC23 S857检测到的靶序列平均密度与uidA 405检测到的平均密度之比约为1：1、243：1和6：1。而rodA 984和EC23 S857检测分类学特异性和灵敏度与uidA 405基因检测相似。由于其多拷贝基因靶标，与uidA 405和rodA 984测定相比，其在大肠杆菌细胞中的表达量更高，因此在通过qPCR方法监测环境沃茨中的这些粪便污染指标时提供了更高的分析灵敏度。
Aims:Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli.Methods and Results:A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxonomic specificity and sensitivity of qPCR assays targeting the rodA gene (rodA984) and two regions of the multi-copy 23S ribosomal RNA gene (EC23S and EC23S857). Experimental analyses of 28 culture collection strains representing E. coli and 21 related non-target species indicated that the uidA405 and rodA984 assays were both 100% specific for E. coli while the EC23S assay was only 29% specific. The EC23S857 assay was only 95% specific due to detection of E. fergusonii. The uidA405, rodA984, EC23S and EC23S857 assays were 85%, 85%, 100% and 86% sensitive, respectively, in detecting 175 presumptive E. coli culture isolates from fresh, marine and waste water samples. In analyses of DNA extracts from 32 fresh, marine and waste water samples, the rodA984, EC23S and EC23S857 assays detected mean densities of target sequences at ratios of approximately 1 : 1, 243 : 1 and 6 : 1 compared with the mean densities detected by the uidA405 assay.Conclusions:The EC23S assay was less specific for E. coli, whereas the rodA984 and EC23S857 assay taxonomic specificities and sensitivities were similar to those of the uidA405 gene assay.Significance and Impact:The EC23S857 assay has a lower limit of detection for E. coli cells than the uidA405 and rodA984 assays due to its multi-copy gene target and therefore provides greater analytical sensitivity in monitoring for these faecal pollution indicators in environmental waters by qPCR methods.