Rapid screening of antimicrobial probiotics using CRISPR cascade

Area of Science:

• Microbiology
• Biotechnology
• Food Science

Background:

• Bacterial infections from animal sources pose a growing threat to human health.
• Rapid and efficient screening methods for antimicrobial agents are crucial for public health and food safety.

Purpose of the Study:

• To develop a nucleic acid amplification-free CRISPR genetic assay for rapid screening of food-origin antimicrobial probiotics.
• To evaluate the assay's performance in detecting and quantifying pathogenic bacteria and assessing antimicrobial effects.

Main Methods:

• A CRISPR-DNAzyme cascade assay (CRISPRzyme assay) was developed, utilizing Cas12a activation and DNAzyme activity for detection.
• The assay eliminates the need for nucleic acid amplification, reducing time and complexity.
• Pathogen quantification in complex samples was performed by end-measurement of fluorescence.

Main Results:

• The CRISPRzyme assay achieved limits of detection of 62 CFU for Vibrio parahaemolyticus, 86 CFU for Salmonella Typhimurium, and 82 CFU for Listeria monocytogenes.
• Isolates of Bacillus and lactic acid bacteria from fermented foods showed significant antimicrobial activity against Vibrio parahaemolyticus.
• Surfactin was identified as the primary antimicrobial component responsible for the observed activity.

Conclusions:

• The CRISPRzyme assay offers a simplified and rapid method for screening antimicrobial probiotics.
• This assay serves as a valuable tool for combating pathogenic bacterial contamination and infection, with applications in food safety and public health.
• The ability to estimate in vivo antimicrobial effects enhances its utility in practical applications.