APPLICATION OF THE POLYMERASE CHAIN-REACTION TECHNIQUE TO THE DETECTION OF PATHOGENS IN WATER

Enteric pathogens may be present in fecally contaminated waters at extremely low concentrations. In addition, these pathogens may be injured when exposed to the environment and may not be able to grow in laboratory culture media or such media may simply not exist for their progagation in the laboratory. It is paramount thus to use techniques which do not depend on culture techniques for the detection of these pathogens and that allow for the detection of single-cell concentrations. The polymerase chain reaction (PCR) technique has been shown to be an excellent and sensitive means of detecting pathogens in waters. Membrane filtration has been combined with PCR and DNA hybridization techniques to be able to detect the DNA equivalent of one single cell in large volumes of water. In addition, this combination of methods allows for the amplification of different target genes that may be present in the sample, since the membrane can be subjected to repeated amplification reactions under different conditions. A Most Probable Number PCR was developed which allows for the quantification of gene copy number and thus permits extrapolation to estimate the number of bacterial cells in the original sample.