title: Highly sensitive and specific detection of viable Escherichia coli in drinking water.
abstract: A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Junhong
authors.initials: J
authors.lastname: Min
authors.forename: Antje J
authors.initials: AJ
authors.lastname: Baeumner
mesh_terms: D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D005784:Gene Amplification; D021141:Nucleic Acid Amplification Techniques; D011786:Quality Control; D015347:RNA Probes; D012329:RNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014876:Water Pollution
chemical_list: D017931:DNA Primers; D015347:RNA Probes; D012329:RNA, Bacterial