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Applied and Environmental Microbiology, June 2008, p. 3831-3838, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02743-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown} ,{dagger}

Robert D. Stedtfeld,1 Samuel W. Baushke,1 Dieter M. Tourlousse,1 Sarah M. Miller,1 Tiffany M. Stedtfeld,1 Erdogan Gulari,4 James M. Tiedje,2,3 and Syed A. Hashsham1,2*

Department of Civil and Environmental Engineering,1 Center for Microbial Ecology,2 Department of Crop and Soil Science, Michigan State University, East Lansing, Michigan 48824,3 Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 481094

Received 5 December 2007/ Accepted 13 April 2008

Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (CT) equation was developed to calculate the number of starting copies based on an experimental CT. The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (R2 = 0.816) was observed between the predicted and experimental CTs based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3' end. The performance of the predictive CT equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive CT equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.

* Corresponding author. Mailing address: Department of Civil and Environmental Engineering, A126 Research Complex-Engineering, 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 18 April 2008.

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

Applied and Environmental Microbiology, June 2008, p. 3831-3838, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02743-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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