Development and validation of an insulated isothermal PCR assay for the rapid detection of Pasteurella multocida

Pasteurella multocida, a ubiquitous opportunistic pathogen, is renowned for causing diverse respiratory tract diseases in animals. In the quest for on-site detection solutions, we developed a rapid insulated isothermal PCR (iiPCR) assay, targeting the sodA gene specific to Pasteurella multocida. This assay demonstrated remarkable specificity by successfully detecting clinical isolates of Pasteurella multocida types A, B, and D, while maintaining negative results for various other pathogens. Its robust sensitivity was evident in detecting positive strain DNA (2.03 copies/mu L) and colonies (1.36x10(3) CFU/mL) as templates, with positive results for undiluted positive samples. The iiPCR method showed exceptional repeatability in triplicate detections of various diluted positive strains. In practical testing, encompassing 188 clinical nasal swab samples from goats, the iiPCR assay performed comparably to a reference PCR, underscoring its accuracy and reliability for on-site Pasteurella multocida detection in veterinary diagnostics.