Agilent Technologies, Santa Clara, California, United States of America.
PLoS One. 2011 Apr 22;6(4):e18967. doi: 10.1371/journal.pone.0018967.
Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers.
多重检测分析方法可以同时分析大量样本集中少量的核酸靶标，这种方法在常规病原体分型、疫情监测和诊断等应用中已经成为首选的检测方法。然而，很少有 DNA 检测平台能够提供一种高通量、敏感且每个测试成本低的中多重分析解决方案。在这项工作中，设计并整合了一种基于 MassCode 技术的增强型基因分型方法，作为高通量中多重分析系统的一部分，该系统有助于对 DNA 靶标进行稳健的定性差异检测。首先，使用带有独特质量标签的引物组进行 MassCode PCR（MC-PCR）对样品进行分析。然后，将 lambda 核酸外切酶和 MassCode 探针阵列与 MC-PCR 产物接触，进行进一步的检测，特异性地识别目标序列。引物和探针的杂交在均相溶液中进行，这与微球或纳米颗粒悬浮阵列相比具有明显的优势。使用台式单四极杆质谱仪以自动化方式检测与所得 MassCode 杂交体偶联的两个同源标签。在开发了一种用于对选定的沙门氏菌血清群和血清型进行亚型分型的 14 重概念验证检测方法后，展示了使用 MassCode 探针阵列进行多重生物分析的应用前景。该 MassCode 系统非常灵活，可以根据需要定制测试面板，包括更多、更少或不同的标记。
