Using DNA microarrays to detect multiple pathogen threats in water

Abstract

We present four studies that illustrate the use of DNA microarrays for the detection and subsequent genotyping of waterborne pathogens. A genotyping array targeting four virulence factor genes in enterohemorrhagic Escherichia coli (EHEC) was tested. The arrays were clearly able to differentiate between E. coli O157:H7 genotypes and E. coli O91:H2. Non-pathogenic E. coli and non-target organisms were not detected on this array. In the second study, an hsp70 gene single nucleotide polymorphism (SNP) array for specific Cryptosporidium parvum detection was constructed to differentiate between principle genotypes. SNPs, and hence differences between genotypes, were easily detected on this type of array. In the third study an array for Helicobacter pylori was tested for simultaneous SNP discrimination and presence or absence of virulence factor genes. Results from this study showed that both SNP discrimination for some conserved genes, and the presence or absence of virulence factor genes was possible. In the fourth 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 the need to perform PCR.