One-pot CRISPR/Cas12b-LAMP platform for dual-mode detection of Pasteurella multocida

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Pasteurella multocida is a major bacterial pathogen impacting public health and causing economic losses.
• Current detection methods for P. multocida are often time-consuming and complex.
• There is a need for simple, accurate, and rapid diagnostic tools for P. multocida.

Purpose of the Study:

• To develop a novel, one-pot detection platform for Pasteurella multocida.
• To combine loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b system for enhanced detection.
• To evaluate the sensitivity, specificity, and clinical applicability of the developed assay.

Main Methods:

• A one-pot assay integrating LAMP and CRISPR/Cas12b was designed.
• Heparin sodium was used to modulate the cis-cleavage activity of Cas12b.
• The assay's performance was assessed using bacterial cultures and spiked milk samples.

Main Results:

• The LAMP-CRISPR/Cas12b assay achieved a detection limit of 5.0 × 10^1 CFU·mL-1.
• The assay demonstrated high specificity, with no cross-reactivity observed with other bacterial species.
• Successful detection of P. multocida was achieved in artificially contaminated milk samples.

Conclusions:

• A simple, accurate, and robust detection system for P. multocida was established.
• The developed LAMP-CRISPR/Cas12b platform shows significant potential for nucleic acid-based diagnostics.
• This assay offers a promising alternative to existing methods for P. multocida detection.