Rapid Detection of Bacillus anthracis in a Microchip-based Real-time PCR Biosensor

Abstract

A miniaturized, fully-automated, PCR-based detection system has been developed for the rapid detection of the pathogenic bacterium Bacillus anthracis. Monolithic silicon DNA purification / real-time PCR chips were fabricated and tested for their ability to purify and detect DNA from bacterial cells. Using silica-coated microstructures and chemical-based lysis, nucleic acids could be isolated, washed and eluted for subsequent real-time PCR. These microstructures were integrated into a detection microchip containing two distinct regions, one for DNA purification and one for real-time PCR. Using an automated detection platform with integrated microprocessor, pumps, valves, thermocycler and fluorescence detector, target bacterial DNA was detected by real-time PCR amplification using SYBR Green fluorescent dye. As few as 40 B. anthracis cells could be detected using this system with an average time for detection of 60 min. Detection was augmented by on-chip melting curve analysis capable of differentiating between positive and false-positive results.