Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays

Abstract

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

Figure 1. Typical results from DH and TSPE-UH suspension microarrays detecting select pathogens.

Two 17-plex bead arrays were developed for the detection of B. anthracis (Ba), F. tularensis (Ft), Y. pestis (Yp), C. burnetii (Cb) and an internal control for DNA extraction and microarray detection (Bt). The microarrays were based on (A) direct hybridization (DH), or (B) target specific primer extension combined with universal microarray hybridization (TSPE-UH) assay formats. Both microarrays make use of identical amplification products from a 16-plex asymmetric PCR. Mean fluorescence intensity (MFI) is displayed for the different probes that are given in Table 1.

Figure 2. DH suspension microarray measurement showing minor cross-reactivity.

Three Y. pestis strains (Yp) and the no template control (NTC) are shown from a DH measurement. Probe isf showed a minor signal when a high load of Y. pestis genomic DNA was amplified.

Figure 3. Detection of mixed pathogens by using DH and TSPE-UH suspension microarrays.

Genomic DNA from B. anthracis (Ba), F. tularensis (Ft), Y. pestis (Yp), C. burnetii (Cb) was mixed in different ratios and measured by using DH (A) and TSPE-UH (B) microarrays. Mean fluorescence intensity (MFI) is displayed for the different pathogen-specific probes (Table 1). The detection of one pathogen is not impeded by the detection of the other targeted pathogens.