Development of first generation in-situ pathogen detection system (Gen1-IPDS) based on NanoGene assay for near real time E. coli O157:H7 detection.

We developed the first generation in-situ pathogen detection system (Gen1-IPDS) based on the NanoGene assay for detecting and quantifying Escherichia coli O157:H7 specific eaeA gene. The NanoGene assay employs the hybridization of target DNA with quantum dot labeled magnetic beads and probe DNAs to detect and quantify the target bacterial gene. The Gen1-IPDS is currently capable of executing four key steps required in the NanoGene assay: sample and reagents introduction, DNA hybridization, magnetic separation of complexes, and sample collection. Operational parameters such as magnet position, hybridization buffer composition, hybridization flow rate, and hybridization temperature were investigated. Using the experimentally determined operational parameters, the target gene was successfully quantified (R(2)=0.97) over a range of six orders of magnitude (10(-12) to 10(-6) mol L(-1)). The limit of detection (LOD) was determined to be 49×10(-15) mol L(-1). The specificity was also demonstrated by the differential discrimination of mismatched target DNAs. The NanoGene assay quantification results via Gen1-IPDS were validated by correlation with its laboratory version (R(2)=0.97).