Detection of Clostridium perfringens Using Novel Methods Based on Recombinase-Aided Amplification Assay-Assisted...

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Clostridium perfringens is a significant pathogen in both human and animal health.
• The need for rapid and sensitive diagnostic tools for C. perfringens is critical for effective disease control and management.
• Existing detection methods may lack the speed or sensitivity required for timely intervention.

Purpose of the Study:

• To develop novel, rapid, and sensitive instrument-free detection methods for Clostridium perfringens.
• To utilize recombinase-aided amplification (RAA) combined with the CRISPR/Cas12a system for enhanced detection capabilities.
• To validate the performance of these new methods against established techniques and diverse sample types.

Main Methods:

• Development of two distinct C. perfringens detection assays: RAA-CRISPR/Cas12a-FL (fluorescence-based) and RAA-CRISPR/Cas12a-LFS (lateral flow strip).
• Assessed the limit of detection (LOD) for both methods using C. perfringens genomic DNA.
• Evaluated specificity against non-target bacteria and compared results with real-time polymerase chain reaction (qPCR) using clinical and spiked samples.

Main Results:

• The RAA-CRISPR/Cas12a-FL assay achieved an LOD of 2 copies, while RAA-CRISPR/Cas12a-LFS had an LOD of 20 copies per reaction.
• Both methods demonstrated 100% consistency with qPCR results and showed no cross-reactivity with other bacterial species.
• The entire detection process for both assays was completed within 1 hour.

Conclusions:

• The developed RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS assays provide rapid, sensitive, and specific detection of C. perfringens.
• These instrument-free methods hold significant potential for early diagnosis of C. perfringens infections in clinical settings.
• The assays offer a promising new diagnostic scheme for both human and veterinary applications.