Design and validation of oligonucleotide primers suitable for waterborne bacterial pathogen detection via real-time qPCR
thesisposted on 24.05.2021, 08:50 authored by Shawn Thomas Clark
Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.