Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering
Wen‐Chih Cheng, Troy A. Horn, Maya Zayats, Georges Rizk, Samuel R. Major, Hongying Zhu, Joseph J. Russell, Zhiguang Xu, Richard E. Rothman, Alfredo Celedon
Abstract
Abstract 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 −1 (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.