Linger Yvonne, Kukhtin Alexander, Golova Julia, Perov Alexander, Qu Peter, Knickerbocker Christopher, Cooney Christopher G, Chandler Darrell P
Akonni Biosystems, Inc.
Akonni Biosystems, Inc.;
J Vis Exp. 2014 Apr 25(86):51256. doi: 10.3791/51256.
Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.
简化微阵列工作流程是创建基于微阵列的耐多药结核病诊断方法的必要第一步，这种诊断方法可在资源较少的环境中常规使用。一种扩增微阵列在单一溶液中并进入单个微流控腔室，将不对称PCR扩增、靶标大小选择、靶标标记和微阵列杂交结合在一起。使用9重不对称主混合物和低密度凝胶元件微阵列对耐多药结核分枝杆菌（MDR-TB）进行基因分型，展示了一种批处理方法。此处描述的方案可在6小时内完成，并能使用至少1000个基因组DNA细胞当量进行正确的基因分型。纳入芯片上的洗涤步骤是可行的，这将产生一种完全封闭的扩增子方法和系统。扩增微阵列的多重程度最终受限于可组合到单个主混合物中并仍能实现所需灵敏度和特异性性能指标的引物对数量，而不是固定在阵列上的探针数量。同样，总分析时间可根据具体预期用途、研究问题和所需检测限缩短或延长。然而，总体方法通过减少人工密集且耗时的处理步骤数量，显著简化了终端用户的微阵列工作流程，并为将基于微阵列的诊断转化为常规临床实践提供了简化的生化和微流控路径。
