Student Research: Jennifer K. Parker

, Environmental Health (EH), 2008
Faculty Advisor: John Meschke

Whole Genome Amplification as a Pre-PCR Step to Improve Detection of Pathogens in Drinking Water


Abstract

Rapid progress has been made in the development of molecular methods for detection of pathogens of public health significance. However, applicability to environmental samples remain limited due to difficulty in recovery and concentration of these pathogens from environmental media. Detection of apthogens in drinking water is of particular concern, as public drinking water utilities supply the majority of the US population, making monitoring of pathogens in drinking water essential. Pathogens n drinking water are highly diffuse, but their existence poses a health risk, as low numbers of pathogens are capable of causing disease. Concentrating these pathogens to a level at which they can be detected from among a variety of background microorganisms is the primary limitation to efficient surviellance. For this reason, indicator organisms are monitored as a surrogate for pathogenic organisms, as they are much simpler to culture and are known to correlate with the presence of fecal material. Indicator organisms, though, are an imperfect measure of the presence of actual pathogens. Whole genome amplification (WGA), a non-specific amplification of all genomic material, may be adapted to drinking water samples to improve molecular detection of pathogens. WGA can serve as a pre-amplification step to increase the levels of DNA prior to PCR detection so that very small quantities of nucleic acids from small amounts of pathogenic organisms can be detected. WGA has shown to amplify small quantities of DNA with minimal amplification bias and increase total sample volume. WGA may help to amplify low levels of pathogens present in diverse microbial communities and also allow more molecular analyses to be performed. Similarly, whole transcriptome amplification (WTA) may be used to improve detection of RNA.

To test the ability of WGA and WTA to improve molecular detection of pathogens in environmental samples, WGA/WTA were applied to mock drinking water samples containing mixed microbial populations spiked with small numbers of pathogens. Drinking water samples designed to represent a typical mixed microbial population were prepared using extracted E. coli and B. subtilis DNA or extracted environmental surface water DNA. These samples were spiked with small quantities of the human pathogens Adenovirus type 41, Aeromonas hydrophila, Mycobacterium avium, and Echovirus type 13. Samples were amplified using a Genomeplex WGA kit and a Transplex WTA kit. Pathogen specific quantitative polymerase chain reaction (qPCR) was used to quantify target pathogens before and after application of WGA/WTA. Copy numbers of target pathogens were greater in samples after application of WGA/WTA. The size of the increase in copy number varied with the amount of background DNA and the amount of the original pathogen spike. Increases in copy number improved subsequent target-specific detection of pathogens. WGA and WTA are able to increase the quantity of target organism DNA in mixed populations. It appears that WGA/WTA can serve as an effective pre-amplification step to increase levels of DNA prior to PCR detection so that nucleic acids from bery small numbers of pathogenic organisms can be more easily detected. Samples amplified by these procedures could be tested for a variety of pathogens of concern, yielding information about human health risks present in drinking water.