An in situ synthesized microfluidic DNA biochip to detect 20 waterborne pathogens
D.M. Tourlousse, A. Munir, R.D. Stedtfeld, F. Ahmad, T. Stedtfeld, A.B. Herzog, J.-M. Rouillard, E. Gulari, J.M. Tiedje and S.A. Hashsham
Michigan State University, US
pathogens, biochip, microfluidic, water, virulence
Parallel and cost-effective detection of pathogens is of interest in many areas including clinical diagnostics, food and water safety, and biodefense. This study reports the development and validation of a microfluidic biochip containing approximately 10,000 in situ synthesized oligonucleotide probes for simultaneous detection of 20 waterborne pathogens. Each pathogen was targeted with 3 to 6 virulence and marker genes. Use of virulence and marker genes as opposed to the 16S rRNA gene used by several previous studies, and use of 5 to 20 oligonucleotide probes per gene are among the key attributes of this biochip. For enhanced sensitivity, multiplex amplification of up to 25 gene targets in a single polymerase chain reaction tube was achieved followed by fluorescent labeling and hybridized to the biochip. Detection limits were estimated to be in the range of 0.01 to 0.001% depending on the GC content of the target gene. Models were developed to assess hybridization time and validated by experimental data. The biochip has the potential to serve as a powerful diagnostic tool for the detection and identification of microorganisms. Considering analysis cost alone, it also provides significant cost savings when samples must be screened for multiple targets ($10-$15 per pathogen).
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Nanotech 2007 Conference Program Abstract