Straub Timothy M, Dockendorff Brian P, Quiñonez-Díaz Maria D, Valdez Catherine O, Shutthanandan Janani I, Tarasevich Barbara J, Grate Jay W, Bruckner-Lea Cynthia J
Interfacial Chemistry and Engineering Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, Mail stop K4-12 Richland, WA 99352, USA.
J Microbiol Methods. 2005 Sep;62(3):303-16. doi: 10.1016/j.mimet.2005.04.012.
Detection of pathogenic microorganisms in environmental samples is a difficult process. Concentration of the organisms of interest also co-concentrates inhibitors of many end-point detection methods, notably, nucleic acid methods. In addition, sensitive, highly multiplexed pathogen detection continues to be problematic. The primary function of the BEADS (Biodetection Enabling Analyte Delivery System) platform is the automated concentration and purification of target analytes from interfering substances, often present in these samples, via a renewable surface column. In one version of BEADS, automated immunomagnetic separation (IMS) is used to separate cells from their samples. Captured cells are transferred to a flow-through thermal cycler where PCR, using labeled primers, is performed. PCR products are then detected by hybridization to a DNA suspension array. In another version of BEADS, cell lysis is performed, and community RNA is purified and directly labeled. Multiplexed detection is accomplished by direct hybridization of the RNA to a planar microarray. The integrated IMS/PCR version of BEADS can successfully purify and amplify 10 E. coli O157:H7 cells from river water samples. Multiplexed PCR assays for the simultaneous detection of E. coli O157:H7, Salmonella, and Shigella on bead suspension arrays was demonstrated for the detection of as few as 100 cells for each organism. Results for the RNA version of BEADS are also showing promising results. Automation yields highly purified RNA, suitable for multiplexed detection on microarrays, with microarray detection specificity equivalent to PCR. Both versions of the BEADS platform show great promise for automated pathogen detection from environmental samples. Highly multiplexed pathogen detection using PCR continues to be problematic, but may be required for trace detection in large volume samples. The RNA approach solves the issues of highly multiplexed PCR and provides "live vs. dead" capabilities. However, sensitivity of the method will need to be improved for RNA analysis to replace PCR.
检测环境样本中的致病微生物是一个困难的过程。对目标微生物进行浓缩时，许多终点检测方法（尤其是核酸方法）的抑制剂也会同时被浓缩。此外，灵敏、高度多重的病原体检测仍然存在问题。BEADS（生物检测促进分析物递送系统）平台的主要功能是通过可再生表面柱，从这些样本中常含有的干扰物质中自动浓缩和纯化目标分析物。在BEADS的一个版本中，利用自动免疫磁珠分离（IMS）从样本中分离细胞。捕获的细胞被转移到流通式热循环仪中，使用标记引物进行PCR。然后通过与DNA悬浮阵列杂交来检测PCR产物。在BEADS的另一个版本中，进行细胞裂解，纯化并直接标记群落RNA。通过将RNA直接与平面微阵列杂交来完成多重检测。BEADS的集成IMS/PCR版本能够成功地从河水样本中纯化并扩增出10个大肠杆菌O157:H7细胞。在磁珠悬浮阵列上同时检测大肠杆菌O157:H7、沙门氏菌和志贺氏菌的多重PCR检测方法已得到验证，每种微生物的检测限低至100个细胞。BEADS的RNA版本的结果也显示出良好前景。自动化操作可产生高度纯化的RNA，适用于微阵列上的多重检测，微阵列检测特异性与PCR相当。BEADS平台的两个版本在从环境样本中自动检测病原体方面都显示出巨大潜力。使用PCR进行高度多重的病原体检测仍然存在问题，但在大量样本的痕量检测中可能是必需的。RNA方法解决了高度多重PCR的问题，并提供了“活细胞与死细胞”区分能力。然而，为了让RNA分析取代PCR，该方法的灵敏度还需要提高。
