Nucleic acid amplification strategies for DNA microarray-based pathogen detection

Abstract

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, phi29 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 phi29 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.

FIG. 1.

Schematic depiction of the methods utilized for specific and random amplification of E. coli O157:H7 genomic DNA for subsequent hybridization to oligonucleotide microarrays.

FIG. 2.

Electrophoretic profiles of specific and random amplification products. Lane 1, unamplified EHEC genomic DNA; lane 2, multiplex PCR; lane 3, random PCR; lane 4, Klenow fragment-based isothermal amplification; lane 5, φ29 polymerase-based isothermal amplification; lane 6, 100-bp ladder. Target gene designations for multiplex PCR products are shown to the left. Twenty percent of each reaction volume was electrophoresed through a precast 4% NuSieve gel.

FIG. 3.

Hybridization of specific and random amplification products to low-density EHEC oligonucleotide microarrays. (Top) Amplification products from both E. coli O157:H7 and E. coli K-12 genomic DNA templates were hybridized to a low-density oligonucleotide microarray with two unique 70-mer probes (1 and 2) per target gene. (A) eaeA; (B) fliC; (C) rfbE; (D) stx1; (E) stx2; (F) ipaH; (G) hexon. The amplification methods used are indicated to the left. The data are from one representative experiment of five performed with independent amplifications. (Bottom) Graphical representation of the hybridization signal intensities obtained from E. coli O157:H7 amplification products. Black bars, row 1 probes; white bars, row 2 probes. The data shown are means + standard deviations from five replicate experiments.