Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
PLoS One. 2012;7(2):e31958. doi: 10.1371/journal.pone.0031958. Epub 2012 Feb 15.
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.
微阵列为同时检测多种病原体提供了一种强大的分析工具。我们开发了诊断悬浮微阵列，用于灵敏和特异性检测生物威胁病原体炭疽杆菌、鼠疫耶尔森菌、土拉弗朗西斯菌和贝氏柯克斯体。开发了两种用于扩增和标记的检测化学方法，一种方法使用直接杂交，另一种方法使用靶标特异性引物延伸，结合与通用微阵列的杂交。使用多重不对称 PCR 扩增 16 个 DNA 特征（16 重），为两种检测化学方法生成了不对称 PCR 产物。比较了两种检测化学方法的性能，并讨论了它们的优缺点。开发的微阵列检测到多个特征序列和内部对照，从而能够自信地识别靶向病原体并评估其毒力潜力。微阵列具有高度特异性，可检测到靶向病原体的各种菌株。与实时 PCR 相比，不同病原体特征的检测限相似或略高。概率分析表明，即使存在少量基因组拷贝，也可以有 95%的置信度进行检测。微阵列可以检测到不同比例混合的不同病原体的 DNA，以及加标或自然污染的样本。开发的检测方法具有在监测和诊断中应用的潜力。
