Improved Detection Techniques for Foodborne Pathogens: Development of Biosensors

Investigator: Rashid Bashir (Department of Bioengineering)

Project Report 2010 - 2011

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Project Rationale

Foodborne pathogens and food safety-related outbreaks have been given increased public and media attention in recent years. From E. coli O157:H7 in spinach to L. monocytogenes outbreaks in ready-to-eat deli meats, consumers are concerned about keeping their food safe. Traditionally, successful amplification of a target gene sequence was confirmed through gel electrophoresis assays, but these were time-consuming analyses. Polymerase chain reaction (PCR) assays for bacterial detection have greatly reduced the time needed for detection. Modern real-time PCR machines utilize double-stranded DNA binding fluorophores, but are expensive and not portable. We have proposed and developed the use of an electrical rather than optical method for conducting PCR assays to help reduce cost and possibly allow the realization of point-of-test PCR devices.

Project Objectives

  • Integrate technology platforms that we have developed, and which have been individually tested in various formats, into a usable technology for detecting L. monocytogenes in less than 8 hours (time to result).
  • Integrate technology platforms, currently being developed, into a usable technology for detecting Salmonella and Shiga toxin-producing E. coli.
  • Develop and optimize methods for simultaneous detection of L. monocytogenes, Salmonella, and Shiga toxin-producing E. coli.
  • Develop microfluidic devices for detection of pathogens using electrical methods for detection of PCR products.

Project Highlights

We applied the label-free impedance spectroscopy method to detection of PCR amplification. We investigated the change in impedance and phase of a solution with changing DNA concentration as DNA molecules were amplified via PCR. To minimize the effects of excess primers, charge shielding, ion binding, and variance in ion composition, the amplified PCR product was first purified to remove primers and PCR reagents and then precipitated out of solution and re-suspended in deionized water to remove excess salt ions. An increase in the relaxation maxima from 0 to 40 cycles in the full PCR sample was observed in the raw data. As confirmed through gel electrophoresis and nanodrop spectrophotometery, the 40 cycle sample contained roughly 1×1011 508 bp molecules/µL. To confirm the increase in relaxation maxima in the phase was solely due to the amplified PCR product, two negative controls, primer only and template only, were also measured.

The amount of target DNA at 40 cycles is consistent with our projected detection limit from the dsDNA in deionized water tests, 1×1011 molecules/µL. From a theoretical standpoint, this level of molecules/µL is possible at 40 cycles when starting from around seven copies of template DNA per 75 µL reaction volume. Also, factoring in reagent limitations from the PCR solution's 200 µM dNTP mix, this experiment's maximum yield was ~1.7×1011 molecules/µL, which is still above our expected detection limit. Hence, this methodology could allow for a detection limit of around 100 CFU/mL. While the current detection limit of this system is relatively high, in the 30-40 cycle range, further development of the system could put its capabilities on par with current real-time PCR detection devices. By combining an on-chip thermocycling process and a PCR purification step with electrical detection of DNA molecules, an on-site diagnostic system with minimal cost and footprint is possible, improving upon current PCR instrumentation. This approach can be used to develop point-of-care devices for detection of pathogenic DNA from bacteria such as L. monocytogenes, Salmonella, E. coli, and others.

""By combining an on-chip thermocycling process and a PCR purification step with electrical detection of DNA molecules, an on-site diagnostic system is possible.""

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