LaGier Michael J, Scholin Christopher A, Fell Jack W, Wang Joseph, Goodwin Kelly D
Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel School for Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA.
Mar Pollut Bull. 2005 Nov;50(11):1251-61. doi: 10.1016/j.marpolbul.2005.04.034.
Monitoring waters for indicator bacteria is required to protect the public from exposure to fecal pollution. Our proof-of-concept study describes a method for detecting fecal coliforms. The coliform Escherichia coli was used as a model fecal indicator. DNA probe-coated magnetic beads in combination with the electrochemical monitoring of the oxidation state of guanine nucleotides should allow for direct detection of bacterial RNA. To demonstrate this concept, we used voltammetry in connection with pencil electrodes to detect isolated E. coli 16S rRNA. Using this approach, 10(7) cells of E. coli were detected in a quantitative, reproducible fashion in 4h. Detection was achieved without a nucleic acid amplification step. The specificity of the assay for coliforms was demonstrated by testing against a panel of bacterial RNA. We also show that E. coli RNA can be detected directly from cell extracts. The method could be used for on-site detection and shows promise for adaptation into automated biosensors for water-quality monitoring.
监测水体中的指示菌对于保护公众免受粪便污染至关重要。我们的概念验证研究描述了一种检测粪大肠菌群的方法。大肠埃希氏菌被用作粪便指示菌的模型。结合鸟嘌呤核苷酸氧化态的电化学监测，DNA探针包被的磁珠应能直接检测细菌RNA。为了证明这一概念，我们使用伏安法结合铅笔电极来检测分离的大肠杆菌16S rRNA。采用这种方法，在4小时内以定量、可重复的方式检测到了10⁷个大肠杆菌细胞。检测无需核酸扩增步骤。通过对一组细菌RNA进行测试，证明了该检测方法对大肠菌群的特异性。我们还表明，大肠杆菌RNA可以直接从细胞提取物中检测到。该方法可用于现场检测，并有望应用于水质监测的自动化生物传感器。
