Molecular approaches to microbiological monitoring: Fecal source detection
Molecular approaches to microbiological monitoring: Fecal source detection
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DOI：
10.1023/a:1021349629950
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发表时间：
2003-01-01
影响因子：
3
通讯作者：
Brodeur, TJ
中科院分区：
文献类型：
--
作者：
Field, KG;Bernhard, AE;Brodeur, TJ
分子方法既可用于监测细菌的自然群落，也可用于在复杂环境中追踪特定的细菌标记。长度异质性聚合酶链反应（LH-PCR）和末端限制性片段长度多态性（T-RFLP）的16 S rRNA基因的16 S rRNA基因的PCR产物的长度多态性的基础上区分。通过这些方法，我们开发了一种替代指标，可以区分水中粪便污染的来源。我们用特异性引物扩增了粪便厌氧拟杆菌属的16 S rRNA基因片段。由于拟杆菌通常居住在肠道栖息地，它在水中的存在表明粪便污染。分子检测规避了厌氧菌生长的复杂性。我们鉴定了反刍动物或人类粪便特有的拟杆菌LH-PCR和T-RFLP核糖体DNA标记物。从受污染的天然沃茨中回收了相同的独特粪便标记物。我们克隆和测序的独特标记，标记序列被用来设计特异性PCR引物，可靠地区分人类和反刍动物的粪便污染源。更多物种的引物正在开发中。这种方法比粪大肠菌群检测更敏感，在复杂性上与标准食品安全和公共卫生诊断测试相当，并且适合自动化和高通量。因此，粪便厌氧菌的分子遗传标记有望用于监测细菌污染和水质。
Molecular methods are useful both to monitor natural communities of bacteria, and to track specific bacterial markers in complex environments. Length-heterogeneity polymerase chain reaction ( LH-PCR) and terminal restriction fragment length polymorphism (T-RFLP) of 16S rDNAs discriminate among 16S rRNA genes based on length polymorphisms of their PCR products. With these methods, we developed an alternative indicator that distinguishes the source of fecal pollution in water. We amplify 16S rRNA gene fragments from the fecal anaerobic genus Bacteroides with specific primers. Because Bacteroides normally resides in gut habitats, its presence in water indicates fecal pollution. Molecular detection circumvents the complexities of growing anaerobic bacteria. We identified Bacteroides LH-PCR and T-RFLP ribosomal DNA markers unique to either ruminant or human feces. The same unique fecal markers were recovered from polluted natural waters. We cloned and sequenced the unique markers; marker sequences were used to design specific PCR primers that reliably distinguish human from ruminant sources of fecal contamination. Primers for more species are under development. This approach is more sensitive than fecal coliform assays, is comparable in complexity to standard food safety and public health diagnostic tests, and lends itself to automation and high-throughput. Thus molecular genetic markers for fecal anaerobic bacteria hold promise for monitoring bacterial pollution and water quality.