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.

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

“…164,168 Other pathogens, including Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Coxiella burnetii were detected in animal and environmental samples using a BBMA. 68 As previously described, the MOL-PCR assay detected 13 signatures of B. anthracis, Y. pestis, and F. tularensis. 40 This methodology was particularly useful because the gene-specific areas detected were short (i.e., DNA targets of 40-50 nucleotides) and could be amenable to detection of degraded targets that can be found in environmental, forensic, and clinical samples.…”

“…40 Such PCR-based BBMAs have been previously described and diagrammed in more detail elsewhere. 68,74,82,102,107 All the aforementioned genotyping BBMAs can be used for host SNP genotyping. As an example, an algorithm for SNP calling using R software was used to assist in SNP association for childhood leukemia patients.…”

Coxiella burnetii - Erreger des Q (query)-Fiebers

“…Zuverlässige PCR-Nachweismethoden für Coxiella sind für die Genotypisierung (s. oben) verwendet worden [8,17,21]. Eine asymmetrische PCR mit Hybridisierung und Analyse der Produkte in Lösung, die gleichzeitig auch weitere Bakterien nachweist, wurde in den Niederlanden entwickelt [124]. Die umfangreichste Charakterisierung von Coxiella-Stämmen erfolgte über die Amplifikation und Sequenzierung von 139 chromosomalen und plasmidischen ORF (open reading frame), einschließlich der Gene für die LPS-Biosynthese [119].…”

Dynamic range extension of hybridization sensors

“…The focus of this manuscript is on hybridization techniques which are appealing due to simplicity: a nucleic acid probe is designed to hybridize to the perfectly matching target molecule with a high efficiency while having a low efficiency for targets 10 containing sequence variations. The principle is open to many technical implementations and miniaturization for use in biosensor devices [1,2,3,4,5,6]. However, hybridization has challenges on the side of probe design and dynamic range due to hybridization stringency conditions [7,8,9] which are not easy to optimize especially when parallelization is aimed for.…”