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Applied and Environmental Microbiology, April 2008, p. 2200-2209, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01962-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water{triangledown} ,{dagger}

Sarah M. Miller,1,{ddagger} Dieter M. Tourlousse,1,{ddagger} Robert D. Stedtfeld,1 Samuel W. Baushke,1 Amanda B. Herzog,1 Lukas M. Wick,3 Jean Marie Rouillard,4 Erdogan Gulari,4 James M. Tiedje,2 and Syed A. Hashsham1,2*

Department of Civil and Environmental Engineering,1 Center for Microbial Ecology,2 National Center for Food Safety and Toxicology, Michigan State University, East Lansing, Michigan 48824,3 Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 481094

Received 27 August 2007/ Accepted 25 January 2008

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately –19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, A126 Research Complex Engineering, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-8241. Fax: (517) 355-0250. E-mail: hashsham{at}egr.msu.edu

{triangledown} Published ahead of print on 1 February 2008.

{ddagger} S.M.M. and D.M.T. contributed equally to this study.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2008, p. 2200-2209, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01962-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Stedtfeld, R. D., Baushke, S. W., Tourlousse, D. M., Miller, S. M., Stedtfeld, T. M., Gulari, E., Tiedje, J. M., Hashsham, S. A. (2008). Development and Experimental Validation of a Predictive Threshold Cycle Equation for Quantification of Virulence and Marker Genes by High-Throughput Nanoliter-Volume PCR on the OpenArray Platform. Appl. Environ. Microbiol. 74: 3831-3838 [Abstract] [Full Text]  


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