A novel typing method for Clostridium perfringens using multiplex recombinase polymerase amplification and...

Area of Science:

• Microbiology
• Molecular Diagnostics
• Bacteriology

Background:

• Clostridium perfringens causes diverse diseases in humans and animals.
• Accurate and rapid typing of C. perfringens is crucial for timely diagnosis and reducing mortality.
• Existing typing methods may lack speed, sensitivity, or accessibility.

Purpose of the Study:

• To develop a rapid, sensitive, accurate, and instrument-free method for C. perfringens typing.
• To target specific toxin genes (α, β, ε, ι) for strain differentiation.
• To provide an improved tool for early disease diagnosis and pathogen identification.

Main Methods:

• Design of specific primer pairs and crRNA sequences for key toxin genes.
• Development of a multiplex recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a system (Cp-MRC12a).
• Validation of the assay using bacterial strains, clinical samples, and spiked samples.

Main Results:

• The Cp-MRC12a assay achieved C. perfringens typing in under 1 hour.
• High sensitivity was demonstrated, with detection limits of 10 copies/μL (type A) and 100 copies/μL (types B-E).
• The assay exhibited high specificity, showing no cross-reactivity with non-target bacteria, and reliable performance on clinical samples.

Conclusions:

• Cp-MRC12a offers a robust and practical approach for C. perfringens typing.
• This method significantly advances early disease diagnosis and pathogen identification.
• The instrument-free nature of Cp-MRC12a enhances its accessibility for various diagnostic settings.