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Applied and Environmental Microbiology, May 2004, p. 3047-3054, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3047-3054.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection

Gary J. Vora,1* Carolyn E. Meador,2 David A. Stenger,1 and Joanne D. Andreadis1,{dagger}

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C. 20375,1 Nova Research, Inc., Alexandria, Virginia 223082

Received 4 September 2003/ Accepted 20 January 2004

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, {phi}29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and {phi}29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

* Corresponding author. Mailing address: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375. Phone: (202) 767-0394. Fax: (202) 404-8688. E-mail: gvora{at}cbmse.nrl.navy.mil.

{dagger} Present address: National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.

Applied and Environmental Microbiology, May 2004, p. 3047-3054, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.3047-3054.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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