Veterinary Research Institute, Department of Food and Feed Safety, Hudcova 296/70, 621 00, Brno, Czech Republic.
Faculty of Science, Department of Botany and Zoology, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
Sci Rep. 2019 Feb 25;9(1):2719. doi: 10.1038/s41598-019-40035-5.
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
多重寡核苷酸连接聚合酶链反应（MOL-PCR）是一种在单个反应中同时检测多个分子标记的快速方法。MOL-PCR 越来越多地应用于微生物检测分析中，其通过简单分析促进鉴定和进一步特征分析的能力具有很大的益处，并大大简化了常规诊断。当适配于 MAGPIX 读取器上的微球悬浮阵列时，MOL-PCR 有可能优于标准的基于核酸的诊断检测方法。本研究通过强调关键参数的适当选择，以食源性病原体（细菌和寄生虫）为例，代表了内部 MOL-PCR 检测方法优化的指导方针。优化后的方案侧重于特定序列检测，利用荧光报告染料 BODIPY-TMRX 和自耦联磁性微球，并允许流畅而轻快的工作流程，这应为开发用于病原体检测的 MOL-PCR 检测方法提供指导。
