CRISPR-Based Detection, Identification and Typing of Mycobacterium tuberculosis Complex Lineages

Area of Science:

• Microbiology
• Genomics
• Molecular Diagnostics

Background:

• Polymerase chain reaction (PCR)-based detection of Mycobacterium tuberculosis complex (MTC) is crucial for diagnosing tuberculosis in clinical microbiology labs.
• Current diagnostic methods require improvement for specificity and lineage identification.

Purpose of the Study:

• To identify a specific gene for MTC detection using genome-wide profiling.
• To develop and validate a novel diagnostic assay for MTC and its lineages.

Main Methods:

• Genome-wide profiling of 3,156 mycobacterial genomes using Roary.
• Real-time PCR (RT-PCR) and PCR-sequencing targeting the identified CRISPR-csm4 gene.
• Validation using MTC and non-tuberculous mycobacteria isolates, and clinical samples.

Main Results:

• The CRISPR-csm4 gene was identified as specific for MTC.
• The CRISPR-csm4 assay accurately detected all 20 MTC isolates and none of the 5 non-tuberculous mycobacteria.
• Clinical sample evaluation showed expected results for all 65 samples, including MTC Beijing lineage identification.

Conclusions:

• CRISPR-csm4-based RT-PCR and PCR-sequencing offer a specific method for MTC detection.
• This assay can complement existing tuberculosis diagnostics, enhancing MTC pathogen identification and lineage differentiation.
• The findings support the implementation of CRISPR-csm4 assays in clinical microbiology laboratories.