A streamlined CRISPR-based test for tuberculosis detection directly from sputum.

Area of Science:

• Molecular Biology
• Diagnostics
• Global Health

Background:

• Tuberculosis (TB) remains a significant global health threat, particularly in underresourced regions.
• There is a critical need for accessible and simple diagnostic tools for TB.
• Current diagnostic methods may be complex or inaccessible in low-resource settings.

Purpose of the Study:

• To develop a rapid, affordable, and simple diagnostic assay for Mycobacterium tuberculosis (Mtb).
• To create a one-pot assay combining amplification and detection for streamlined TB diagnosis.
• To ensure the assay is compatible with lateral flow and lyophilization for field use.

Main Methods:

• Utilized recombinase polymerase amplification (RPA) combined with Cas13a and Cas12a detection.
• Developed two parallel one-pot assays to detect Mtb-specific elements (IS6110, IS1081) and a human DNA control.
• Validated the assay using spiked sputum samples and clinical blinded sputum samples.

Main Results:

• Achieved a limit of detection of 69.0 CFU/mL for Mtb H37Rv and 80.5 CFU/mL for Mycobacterium bovis BCG.
• Demonstrated no cross-reactivity with other bacterial or fungal isolates.
• Clinical testing showed 100% sensitivity and 100% specificity compared to culture in blinded samples.

Conclusions:

• The SHINE-TB assay effectively streamlines TB diagnosis from sample to result.
• The assay's compatibility with lateral flow and lyophilization makes it suitable for underresourced areas.
• This innovative diagnostic approach holds promise for improving global TB control efforts.