Rapid on-site detection of Pasteurella multocida in cattle using an LFS-based RPA-CRISPR/Cas12a assay

Pasteurella multocida is an important bacterial pathogen associated with bovine respiratory disease (BRD), causing substantial economic losses in cattle production worldwide. Rapid field detection of P. multocida remains challenging because conventional diagnostic methods are often time-consuming and require specialized laboratory equipment. This study aimed to establish a rapid visual assay for P. multocida to support field diagnosis and disease management in cattle.

A recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the species-specific kmt1 gene of P. multocida was developed and combined with lateral flow strips (LFS) for instrument-free visual detection. The assay performance was comprehensively evaluated based on sensitivity, specificity, repeatability, and validation using clinical samples.

Under optimized conditions, the RPA amplification was completed within 15 min at 37 °C, and the CRISPR/Cas12a reaction generated clear visual signals on the LFS. The assay achieved a detection limit of 10¹ copies/µL, showing higher sensitivity compared with conventional PCR. No cross-reactivity was observed with ten common bovine pathogens, indicating high specificity. Repeatability testing demonstrated stable and consistent performance. Among 40 clinical bovine samples, the assay results were fully consistent with conventional PCR, successfully identifying 17 positive and 23 negative samples.

The established LFS-based RPA-CRISPR/Cas12a assay enables rapid, sensitive, and instrument-independent detection of P. multocida. This method provides a practical point-of-care testing (POCT) tool for rapid on-site screening of P. multocida in resource-limited veterinary settings.