Rapid and Visual Detection of Pasteurella multocida and Riemerella anatipestifer Using Recombinase Polymerase...

To establish a convenient, novel, and visual detection method for on-site diagnosis of Riemerella anatipestifer (RA) and Pasteurella multocida (PM), we used the previously constructed plasmids pMD19-T-ompA and pMD19-T-kmt1 as templates and selected as target sequences the conserved regions of the RA ompA gene sequence and the PM kmt1 gene sequence registered in GenBank. Specific basic primers for recombinase polymerase amplification (RPA) were designed and synthesized. Probes (RA-P, PM-P) were designed according to the requirements of the lateral flow dipstick (LFD) assay. After optimizing the reaction conditions, a dual RPA-LFD method for detecting RA and PM was established, and specificity and sensitivity tests were conducted. The developed method was then applied to 64 clinical samples. The established dual RPA-LFD method completed amplification within 15 min at 37 C. Using the nucleic acids of Escherichia coli from ducks, Salmonella enteritidis from ducks, RA and PM from poultry, Staphylococcus, goose parvovirus, duck plague virus, and Muscovy duck parvovirus as templates, and plasmids pMD19-T-ompA and pMD19-T-kmt1 as positive controls, the dual RPA-LFD detection system showed that only the positive controls yielded positive results, indicating good specificity. Plasmid standards pMD19-T-ompA and pMD19-T-kmt1 were diluted tenfold, and the sensitivity was detected to be 10² copies/µl when plasmid standards with concentrations ranging from 10⁷ to 10⁰ copies/µl were used as templates. For the 64 clinical samples, results were observed directly via the test strips, showing an RA detection rate of 10.93% (7/64) and a PM detection rate of 4.68% (3/64), with a 100% concordance rate with PCR results. The dual RPA-LFD method successfully established in this study has the advantages of strong specificity, high sensitivity, rapidity, and visualization, making it suitable for the on-site detection of RA and PM.