On-Chip PCR, Nanoparticles, and Virulence/Marker Genes for Simultaneous Detection of 20 Waterborne Pathogens and Potential Indicator OrganismsEPA Grant Number: R833010
Title: On-Chip PCR, Nanoparticles, and Virulence/Marker Genes for Simultaneous Detection of 20 Waterborne Pathogens and Potential Indicator Organisms
Investigators: Hashsham, Syed
Current Investigators: Hashsham, Syed , Tiedje, James M. , Tarabara, Volodymyr
Institution: Michigan State University
EPA Project Officer: Page, Angela
Project Period: September 1, 2006 through August 31, 2009 (Extended to August 31, 2011)
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
The objective of this research is to develop and validate a highly parallel, sensitive, specific, and quantitative biochip combining the principles of polymerase chain reaction (PCR) and microarrays for the simultaneous detection of 20 waterborne pathogens. Establishing highly parallel and specific methods are essential to reduce the health risk from microbial pathogens present in source and drinking waters.
Over 250 virulence and marker genes serving as signatures for 20 selected pathogens and 30 potential indicator organisms will be assayed in parallel using a novel on-chip PCR assay. The on-chip PCR device will be capable of amplifying multiple targets and samples with a high level of sensitivity, specificity, and quantitation. A highly efficient cross flow microfiltration process for sample concentration will also be incorporated to provide the sample processing step from surface water to the chip and add to the sensitivity. The list of 20 pathogens includes three bacterial candidates included in the Environmental Protection Agency’s contaminant candidate list (Aeromonas hydrophila, Helicobacter pylori, and Mycobacterium avium intercellulare) as well as 17 other organisms including Cryptosporidium and Giardia. The detection method will be validated by spiking various concentrations of relevant organisms in a number of source and treated drinking water samples. The method will also target selected marker genes from potential indicator organisms. The proposed method is expected to have an overall detection limit of 1 target cell per 100 mL of source or drinking water, be performed in less than 4 hours, and employ a quantitative strategy similar to real time PCR. A small part of the project is also devoted to developing viability assays using a nano-particle based technology capable of detecting a change in bacterial cell concentration of 10 to 100 cells in less than 1 hour.
The developed method will provide a high throughput tool capable of quantitatively monitoring multiple waterborne pathogens using their signature virulence and marker genes. The method when developed can be used as an economical tool to screen for many pathogens with high specificity and sensitivity in the water and wastewater industry.