Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.

► Low-copy targets offered increased specificity for pathogen detection.
► Sensitivities equivalent to minimum infectious doses were defined for test strains.
► Membrane filtration can be applied to reduce extracellular DNA and avoid enrichment.