Use of a recombinase polymerase amplification commercial kit for rapid visual detection of Pasteurella multocida

Area of Science:

• Veterinary Microbiology
• Molecular Diagnostics
• Animal Health

Background:

• Pasteurella multocida (P. multocida) causes significant diseases like bovine respiratory disease (BRD) and haemorrhagic septicaemia (HS) in cattle, buffaloes, and bison.
• Early detection and screening of infected animals are crucial for managing Pasteurellosis.
• Currently, there are no reported methods combining recombinase polymerase amplification (RPA) with lateral flow dipstick (LFD) for P. multocida detection.

Purpose of the Study:

• To develop and evaluate a novel isothermal detection method for P. multocida.
• To assess the potential of combining RPA with LFD for rapid, on-site diagnosis of Pasteurellosis.

Main Methods:

• Development of an isothermal recombinase polymerase amplification (RPA) assay targeting P. multocida DNA.
• Integration of the RPA assay with a lateral flow dipstick (LFD) for visual detection.
• Analytical validation of sensitivity and specificity.
• Clinical evaluation using nasal swabs and lung samples from dairy farms, compared against real-time quantitative PCR (qPCR) and culture methods.

Main Results:

• The P. multocida RPA-LFD assay detected DNA within 30 minutes at 39°C with a detection limit of 120 copies per reaction.
• Clinical evaluation showed 95.15% specificity and a 0.958 kappa coefficient compared to qPCR.
• Compared to culture, the RPA-LFD assay achieved 100% sensitivity and a 0.572 kappa coefficient.

Conclusions:

• The developed RPA-LFD assay is effective and practical for detecting P. multocida.
• The method holds significant potential for development into a routine on-site diagnostic protocol for Pasteurellosis.
• This approach offers a rapid and accessible tool for animal health management.