Ultra-sensitive and rapid detection of nucleic acids and microorganisms in body fluids using single-molecule tethering

Wen-Chih Cheng,Hongying Zhu,Maya Zayats,Richard E Rothman,Zhiguang Xu,Troy Horn,Samuel Major,Joseph Russell,Alfredo Celedon,Georges Rizk

doi:10.1038/s41467-020-18574-7

Wen-Chih Cheng, Hongying Zhu + Show 8 more

Open Access

https://doi.org/10.1038/s41467-020-18574-7

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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.

Highlights

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases
The displacement of beads located in a large area (24 mm2) is determined with submicrometer precision by processing images obtained with a lowmagnification lens and a low-cost digital camera (See “Methods” and Supplementary Figs. 1, 2)
This highly denaturing environment prevents aggregation and inactivates nucleases that otherwise would degrade target molecules and probes. We find that this simple step is sufficient to lyse the tested microorganisms without the need for bead beating nor enzymes

Summary

Introduction

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. 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. Polymerase-based methodologies require complex sample preparation steps to remove polymerase inhibitors in certain specimen types, and relatively expensive reagents and instrumentation[2,3] These limitations have complicated the development and implementation of PCR-based systems for some applications, such as culture-free sepsis diagnosis. Some examples include nanostructured microelectrode[11], bio-bar-code[12], single-molecule array[13], electrochemical biosensor[14], and surface plasmon resonance sensor[15] Many of these methods are time-consuming and have shown insufficient sensitivity for detection of microorganisms, with LODs between 103 and 107 CFU mL−1 11,13,14. We show that SMOLT detects microbes directly in whole-blood samples with LODs between 1 and 3 CFU mL−1

Methods

Results

Conclusion