Rapid specific detection of oral bacteria using Cas13-based SHERLOCK

Area of Science:

• Molecular Biology
• Microbiology
• Biotechnology

Background:

• Oral microbial communities are implicated in diseases like periodontitis and caries.
• Current detection methods (qPCR, NGS) are costly, slow, and complex for widespread screening.
• A need exists for rapid, low-cost oral bacteria detection at the point-of-care.

Purpose of the Study:

• To tailor the CRISPR-Cas based SHERLOCK assay for species-specific oral bacteria detection.
• To develop a computational pipeline for generating SHERLOCK constructs.
• To validate SHERLOCK's performance directly on unprocessed saliva samples.

Main Methods:

• Developed a computational pipeline for designing SHERLOCK assay constructs.
• Experimentally validated species-specific detection of seven oral bacteria.
• Tested assay performance on unprocessed human saliva samples, comparing with 16S rRNA sequencing.

Main Results:

• Achieved single-molecule level detection of oral bacteria with high specificity, even in complex saliva matrices.
• Adapted SHERLOCK for direct detection from unprocessed saliva samples.
• Detection results from 30 healthy saliva samples correlated perfectly with 16S rRNA sequencing.

Conclusions:

• The SHERLOCK assay provides a rapid, low-cost, and specific method for oral bacteria detection.
• This CRISPR-based approach is highly scalable and suitable for point-of-care applications.
• Enables improved diagnostics and screening for oral microbiome-associated diseases.