Rapid Detection of Bacillus anthracis in a Microchip-based Real-time PCR Biosensor
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0952-F05-01
Rapid Detection of Bacillus Anthracis in a Microchip-Based Real-Time PCR Biosensor Nathaniel Charles Cady1, Scott J. Stelick2, and Carl Batt2 1 College of Nanoscale Science & Engineering, University at Albany, 255 Fuller Road, Albany, NY, 12203 2 Food Science, Cornell University, 318 Stocking Hall, Ithaca, NY, 14853
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. INTRODUCTION In the past decade there has been an increased demand for rapid and accurate methods of detecting pathogenic bacteria, viruses and other disease-causing agents. In response to these demands, biosensors have been developed utilizing a variety of existing semiconductor processing strategies. The resulting devices, collectively known as lab-on-a-chip devices, incorporate multiple laboratory processes in a semi-automated, miniaturized format. Many of these devices utilize the polymerase chain reaction (PCR) which is relatively robust, however, a variety of contaminants can inhibit amplification and diminish the success of such analytical instruments [1]. In order to circumvent this problem, DNA must be extracted and purified from a sample through a variety of lysis protocols and purification techniques [2]. In a previous study, our laboratory demonstrated effective bacterial cell lysis and DNA purification in a microfabricated silicon chip [3, 4]. In order to extend the utility of chip-based DNA purification, we have developed an integrated DNA purification and real-time PCR based biosensor that is the subject of the research presented here. Successful chip-based DNA purification and PCR requires not only manufacturing of the detection microchips, but also development of a platform to perform the necessary thermal cycling, fluorescent measurement and fluid control systems. We report here on the construction of a miniaturized lab-on-a-chip for the detection of the pathogen Bacillus anthracis. Because this organism is a current threat as a biowarfare agent, it is an ideal organism to use for model studies of the miniaturized detectio
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