Digital PCR: a tool in clostridial mutant selection and detection

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The ClosTron mutagenesis system facilitates clostridial genome editing.
• Validation of site-specific insertions is crucial to avoid errors.
• Conventional PCR confirms on-target insertions but struggles with off-target detection.

Purpose of the Study:

• To introduce digital PCR (dPCR) as an alternative validation tool for ClosTron mutagenesis in clostridia.
• To assess dPCR's efficacy in selecting accurate mutant strains and quantifying genetic elements.
• To explore dPCR's potential in analyzing complex microbial samples.

Main Methods:

• Construction of Clostridium perfringens chitinase mutant strains using ClosTron.
• Validation of on-target intron insertion using conventional PCR.
• Quantification of ClosTron intron and a reference gene using dPCR to detect off-target insertions.

Main Results:

• dPCR accurately quantifies the mobile intron and reference gene, enabling selection of strains without off-target insertions.
• Mutant strains with equivalent gene amounts were confirmed to lack additional mobile ClosTron inserts.
• dPCR proved effective in distinguishing wildtype and mutant C. perfringens in faecal samples, overcoming limitations of plating and qPCR.

Conclusions:

• Digital PCR is a high-throughput and accurate method for selecting clostridial insertion mutants.
• dPCR serves as a robust tool for quantifying bacterial strains in complex matrices.
• dPCR shows significant potential for broader applications in microbiological research.