Quantitative PCR Combined with Constant Denaturant Capillary Electrophoresis for the Analysis of Naturally Occurring Pathogens in Coastal Environments

Objectives:To develop and apply molecular techniques that allow the quantification of diversity and abundance of pathogenic vibrios in marine environmental sampels. To design and test ribosomal DNA specific primers for the identification of specific vibrios in complex communities. To adapt and optimize Constant Denaturant Capillary Electrophoresis (CDCE) for the separation and quantification of specific amplification products. To develop a competitive, quantitative PCR protocol for the enumeration of the dominant vibrio species in coastal environments and for the determination of vibrio diversity. To test these protocols in environmental samples.Methodology:To date, identification and enumeration of pathogens in the environment is still largely carried out by culturing the bacteria in specific media. However in the last few years it has become evident that culture-based methods are potentially biased and that many pathogens can enter a state during which they are not culturable but remain viable. These problems can be circumvented by the application of molecular techniques that area potentially less biased and more efficient means of identifying and quantifying pathogenic species in the environment. Vibrio species were chosen as a model system as these bacteria are already well studied; however, we anticipate that the protocols will be generally applicable to the enumeration of indicator species, introduced pathogens (e.g. from sewage outfall) or natural occurring harmful microorganisms.Rationale:In quantifying pathogenic organisms in the environment, high sensitivity, accuracy and resolution are crucial to be able to interpret patterns of pathogen transmission or outbreak and to identify environmental refuges. Thus, we have chosen a combination of competitive quantitative PCR (QPCR) and Constant Denaturant Capillary Electrophoresis (CDCE). QPCR allows the controlled amplification of specific targets while CDCE provides for a very accurate and sensitive quantification of amplification products. Both techniques are established in our laboratory and will be adapted in an exemplary fashion to pathogen detection in environmental samples.