Quantitative detection of Staphylococcus aureus by TaqMan real time PCR, using a DNA-based microfluidic biosensor.

A portable integrated detection platform was used for the rapid identification and quantification of S. aureus, one of the most common bacterial pathogens responsible for food poisoning worldwide. In particular, monolithic DNA purification/real time PCR silicon microchips were fabricated and tested for their ability to purify and quantitatively detect S. aureus DNA by TaqMan real-time PCR targeting the thermonuclease (nuc) gene. Microchips were used in conjunction with an automated detection platform integrating a microprocessor, pumps, valves, thermocycler and fluorescence detection modules. The resulting miniaturized, fully automated and integrated detection system was assayed for its sensitivity against serial dilutions of either S. aureus broth culture or S. aureus pre-purified DNA. As few as 1 pg of prepurified DNA (approximately 102 S. aureus genome equivalents) as well as 105 cells of the pathogenic microorganism could be detected with an average time for detection (DNA extraction/real time PCR amplification) of only 40 min. While ongoing work is focusing on improving the detection limit this is one of the first fully automated, miniaturized detection systems for integrated sample preparation and detection of pathogenic bacteria. Acknowledgments: Financial support was provided by the Alliance for Nanomedical Technologies, USDA Grant #03-35201-13691 and FDA Grant #06000002499A. This work was performed in part at the Cornell NanoScale Science & Technology Facility which is supported by the National Science Foundation under Grant ECS-9731293, its users, Cornell University and Industrial Affiliates.