A Streamlined Point-of-Care CRISPR Test for Tuberculosis Detection Directly from Sputum.

Area of Science:

• Molecular Biology
• Diagnostics
• Global Health

Background:

• Tuberculosis (TB) remains a significant global health threat, causing over a million deaths annually.
• There is a critical need for accessible, low-cost diagnostic tools for TB, especially in resource-limited settings.
• Current diagnostic methods can be slow or require specialized equipment, hindering timely treatment.

Purpose of the Study:

• To develop a simple, point-of-care CRISPR-based isothermal assay for diagnosing Mycobacterium tuberculosis (Mtb).
• To integrate a straightforward sample processing technique with the assay for ease of use in under-resourced areas.

Main Methods:

• Combined Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a enzymes in parallel one-pot assays.
• Developed assays to detect two conserved Mtb elements (IS6110 and IS1081) and an internal human DNA control.
• Validated assay compatibility with lateral flow detection and lyophilization for stability.

Main Results:

• The assay demonstrated sensitivity across a broad range of bacterial loads (10^2 to 10^5 CFU/mL) in sputum samples.
• Achieved a limit of detection of 69.0 CFU/mL for Mtb H37Rv and 80.5 CFU/mL for M. bovis BCG.
• Clinical testing showed 100% sensitivity and specificity compared to TB culture, comparable to GeneXpert MTB/RIF Ultra.

Conclusions:

• The developed CRISPR-based assay provides a rapid, robust, and point-of-care solution for TB diagnosis.
• The assay's compatibility with lateral flow and lyophilization enhances its utility in resource-limited settings.
• This integrated approach streamlines TB diagnosis from sample to result, offering a significant advancement.