Scanogen Inc., Baltimore, MD, 21244, USA.
Department of Emergency Medicine, Johns Hopkins University, Baltimore, MD, 21209, USA.
Nat Commun. 2020 Sep 22;11(1):4774. doi: 10.1038/s41467-020-18574-7.
Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.
体液中微生物核酸的检测已成为许多传染病快速诊断的首选方法。然而，在某些情况下，如脓毒症，基于培养的诊断仍然是金标准方法。因此，迫切需要新的无培养方法。在这里，我们描述了一种无扩增和无纯化的分子检测方法，即单分子系链（SMOLT），该方法可以在无需培养的情况下，以高灵敏度检测体液中的微生物。SMOLT 的信号是通过 DNA 探针连接的微米大小的珠子的置换产生的，这些 DNA 探针的长度在 1 到 7 微米之间。使用强大且快速的光学方法，可以以亚微米的精度确定数千个 DNA 探针的分子延伸。我们证明，SMOLT 可以在血液、尿液和痰液中以亚皮摩尔浓度直接检测核酸，并且在血液中以 1 CFU mL（每毫升形成菌落单位）的速度快三倍，具有更高的多重检测能力和比扩增方法更直接的方案。SMOLT 的临床应用价值进一步通过开发一种用于直接在全血中同时检测导致脓毒症的念珠菌属的多重检测方法得到证明。
