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.

Ab Initio and in Situ Comparison of Caffeine, Triclosan, and Triclocarban as Indicators of Sewage-Derived Microbes in Surface Waters

“…Potential markers of wastewater leakage to surface water can be classified as microbial, biomolecular, or chemical. Bacteria, coliphages, mammalian viruses, and protozoa typically are enumerated using culturing techniques, including selective plate counts – , or biomolecular techniques that target nucleic acids with the polymerase chain reaction or proteins with enzymatic and immunological assays – . Organic wastewater compounds (OWCs), including fecal steroids, caffeine, pharmaceuticals, and antimicrobial consumer product additives, are chemical markers that can be tracked by gas or liquid chromatography/mass spectrometry and tandem mass spectrometry ,, .…”

“…Microbial assays relying on culturing techniques typically take from 18 to 96 h , , whereas biomolecular and chemical analyses can be completed in hours or even minutes , . Targeting chemical markers is beneficial since many microbial indicator organisms extant in raw sewage also are common to wildlife and sources other than wastewater , . In addition, chemical markers consistently occur at the highest levels at the source and at relatively lower concentrations in receiving streams , , whereas microbial indicators potentially can multiply in surface waters or become temporally inactive, i.e., viable but nonculturable .…”