AWWA PATH56519 ✓ Most Recent

Methods for the detection of waterborne pathogens often focus on a single disease-causing organism or related group of organisms (e.g. Cryptosporidium parvum or coliforms) at the exclusion of other potential pathogens in water. Thus, there has been a proliferation of methods to detect every single pathogen threat that may be present in the watershed. Often, each of these methods requires significant sample manipulation, a high degree of technical expertise, and are time consuming. To overcome these problems, several researchers have investigated integrated sample collection, sample concentration, and purification systems. However, there is no published method that employs an integrated detection system. This means that the remaining processed sample still requires multiple assays to find all pathogens of interest. Nucleic acid microarrays, or DNA chips, represent the latest advance in molecular technology by providing unparalleled opportunities for multiplexed detection of nucleic acids. With the ability to array 1,000s of genes on a single glass microscope slide, and new sensitive methods to label community DNA or RNA directly, without the use of PCR, microarrays can provide multiplexed detection of all pathogens, indicators, and even their genotypes in a water sample. This paper presents 4 studies that illustrate the potential use of DNA microarrays for the detection and subsequent genotyping of waterborne pathogens. For the first study a genotyping array for pathogenic E. coli was tested. The arrays were clearly able to differentiate between different E. coli O157:H7 genotypes, E. coli O91:H2, and non-pathogenic E. coli. In the second study, a single nucleotide polymorphism array was constructed based on the hsp70 sequence of Cryptosporidium parvum. Both the differences between genotypes and SNP detection were achieved. In the 3rd study, an array for Helicobacter pylori was constructed to simultaneously determine the presence or absence of virulence factor genes and SNP detection. This array was able to distinguish between isolates lacking virulence factor genes, and could provide SNP level detection for conserved genes. In the 4th study, multiplexing was achieved by direct hybridization and detection of mRNA to the array. For highly expressed genes, visible signal was detected at 312.5 ng of total RNA, indicating that these new methods may have sufficient environmental sensitivity without invoking PCR. The paper discusses the goal of working towards an integrated sample collection, sample processing, and detection system that will use a single method to detect all pathogens that may be present in a water sample. The features of this system are: it will be able to handle large volumes of water; be able to further concentrate, lyse, purify, and label total nucleic acids; and, use an array based detector to determine the types of pathogens that may be present in a water sample. Includes 19 references, figures. See more