Optimized DNA microarray assay allows detection and genotyping of single PCR-amplifiable target copies

Abstract

This study was conducted to determine the detection limit of an optimized DNA microarray assay for detection and species identification of chlamydiae. Examination of dilution series of a plasmid standard carrying the target sequence from Chlamydia trachomatis and genomic DNA of this organism revealed that a single PCR-amplifiable target copy was sufficient to obtain a specific hybridization pattern. This performance renders the test suitable for routine testing of clinical samples.

Introduction

While DNA microarray technology has been widely used in gene expression monitoring, genotyping has emerged as another area of application in the last few years. The highly parallel approach, i.e. the possibility to obtain precise sequence information on a variety of genomic loci renders DNA microarrays promising diagnostic tools. Given the complex nature of many bacterial virulence factors, the current PCR-based methods are not capable to fulfill the criteria required for highly informative diagnostic tests in the future. Multi-locus genotyping assays or even genomotyping [1] will supplant the ‘one-dimensional’ typing methods used nowadays.

Recent applications of DNA microarrays in genotyping include detection of antibiotic resistance genes in gram-positive bacteria [2], [3], toxin typing of Clostridium perfringens [4], species differentiation among mixed bacterial communities [5], and identification of respiratory pathogens [6]. However, the suitability of DNA microarray assays for routine diagnosis has yet to be demonstrated as most studies have been dealing with bacterial cultures rather than direct examination of clinical specimens. To achieve this goal, any such test should be easy-to-handle and cost efficient, as well as highly sensitive and specific.

The recently developed ArrayTube™ (AT) platform represents an interesting alternative to the widely used, but relatively expensive fluorescence-based glass slide microarray systems. It involves chips of 2.4×2.4 mm size placed on the bottom of 1.5-ml plastic micro-reaction tubes. Hybridization can be conducted on standard laboratory equipment without changing vessels. In a previous paper, we described the development of an AT microarray to differentiate among all nine species of Chlamydia (C.) and Chlamydophila [7]. In the present study, an optimized protocol of this assay was examined to determine detection limits and identify factors limiting sensitivity.