A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations...

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• CRISPR-Cas13a systems offer rapid and sensitive DNA detection.
• A clear screening strategy for crRNAs for single-base resolution DNA detection of pathogens is needed.

Purpose of the Study:

• To develop and validate a crRNA screening strategy for CRISPR-Cas13a-based single-nucleotide polymorphism (SNP) detection.
• To assess the system's ability to detect drug resistance mutations in Mycobacterium tuberculosis.

Main Methods:

• A combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was employed.
• The CRISPR-Cas13a system was optimized using a set of crRNAs to distinguish specific mutations.

Main Results:

• The developed crRNA set enabled significant discrimination between wild-type and fluoroquinolone resistance mutations in Mycobacterium tuberculosis strains (signal ratio 8.29–38.22).
• Clinical evaluation using genomic DNA showed 100% specificity and 91.4% sensitivity compared to culture-based assays.

Conclusions:

• The optimized CRISPR-Cas13a system demonstrates potential for accurate SNP detection, particularly for identifying drug resistance mutations.
• The proposed crRNA screening strategy is valuable for early drug resistance monitoring and clinical treatment guidance.