We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.

Environmental control of growth and persistence of vibrios in aquatic environments is poorly understood even though members of the genus Vibrio are globally important pathogens. To study how algal-derived organic matter and temperature influenced the abundance of different Vibrio spp., Baltic Sea microcosms inoculated with Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and native bacterioplankton, were exposed to different temperatures (12-25 degrees C) and amended with dissolved organic matter from Nodularia spumigena (0-4.2 mg C L(-1)). Vibrio abundance was monitored by culture-dependent and molecular methods. Results suggested that Vibrio populations entered a viable but nonculturable state during the incubations. Abundance of Vibrio spp. and total bacterioplankton were orders of magnitude higher in microcosms amended with organic matter compared with reference microcosms. Vibrio cholerae abundances ranged from 0.9 to 1.9 x 10(5) cells mL(-1) in treatments amended with 4.2 mg C L(-1). Vibrio cholerae abundance relative to total bacterioplankton and other Vibrio spp. also increased >10-fold. In addition, V. vulnificus abundance increased in mesocosms with the highest organic matter addition (0.9-1.8 x 10(4) cells mL(-1)). Temperature alone did not significantly affect abundances of total bacterioplankton, total Vibrio spp. or individual Vibrio populations. By contrast, cyanobacterial-derived organic matter represented an important factor regulating growth and abundance of V. cholerae and V. vulnificus in brackish waters.

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Twenty-five strains of Plesiomonas shigelloides isolated from aquatic environment, 10 strains from human cases of diarrhoea and five strains from animals were identified by the polymerase chain reaction technique based on 23S rRNA gene. For this purpose, two primers targeted against part of the 5' half of the 23S rRNA gene of P. shigelloides (Escherichia coli number C-912, G-1195; Plesiomonas number C-906, G-1189) were designed. Results from our study indicated that this method might serve as a tool for a rapid and sensitive identification of P. shigelloides from different environmental and clinical sources.