A microchip-based DNA purification and real-time PCR biosensor for bacterial detection

Abstract

A miniaturized, fully-automated, PCR (polymerase chain reaction)-based detection system has been developed for the rapid detection of bacteria. Monolithic DNA purification/real-time PCR silicon chips were fabricated and tested for their ability to purify and detect the pathogenic bacteria Salmonella typhimurium. Using silica-coated microstructures, nucleic acids could be selectively bound, 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 detection modules, microchips were used to purify and detect bacterial DNA by real-time PCR amplification using SYBR Green fluorescent dye. As few as 2/spl times/10/sup 3/ S. typhimurium cells could be detected using this system with an average time for detection of 45 min. Detection was augmented by on-chip melting curve analysis capable of differentiating between positive and false-positive results.