Establishment and application of multiplex PCR assay for bovine pasteurellosis

Abstract:

[Background] Bovine pasteurellosis, caused by serotypes A, B, and E of Pasteurella multocida (Pm), is an acute infectious disease of cattle. Polymerase chain reaction (PCR) is an effective means to diagnose and control this disease. [Objective] To establish a multiplex PCR assay for rapid detection of serotypes A, B, and E of Pm and provide technical support for the rapid and accurate clinical diagnosis of bovine pasteurellosis. [Methods] According to the conserved regions of hyaD-hyaC, bcbD, and ecbJ genes of Pm, we designed three pairs of specific primers for PCR and determined the appropriate annealing temperature (T_m) by temperature-gradient PCR assay. Chessboard test was employed to optimize the primer concentration and then a multiplex PCR assay was established. The recombinant plasmid standard and positive strain were used to determine the sensitivity (limit of detection) of the established assay. The nucleic acid samples of 8 common bovine pathogens (Mannheimia hemolytica C1655, Escherichia coli C237, Listeria monocytogenes C1597, Staphylococcus aureus C3053, Salmonella dublin C79351, Mycobacterium paratuberculosis C1625, bovine infectious rhinotracheitis virus CAV1546, and Mycoplasma bovis C65-1) were used to determine the specificity of the multiplex PCR assay. Three batches of diagnostic reagents were prepared to perform inter-batch and intra-batch tests on sensitive and specific samples to determine the repeatability of the assay. Three different models of PCR instruments were used to detect sensitive and specific samples with the established method to determine the applicability of the assay. The performance of the assay in clinical application was evaluated by detection of clinical samples and simulated infection samples. [Results] The optimal multiplex PCR assay was established under the following conditions: T_m of 55 ℃ and the three pairs of primers at the concentrations of 0.25, 0.30, and 0.20 μmol/L, respectively. The established method could simultaneously detect Pm serotypes A (821 bp), B (203 bp), and E (363 bp). The multiplex PCR assay had high sensitivity. It showed the limits of detection of 43.080, 3.710, and 4.350 copies/μL for recombinant plasmid standards pMD-A, pMD-B, and pMD-E, respectively, as well as the limit of detection of 10² CFU for the positive bacterial liquid. Moreover, the established assay had strong specificity as it only produced bands for the serotypes A, B, and E of Pm and no bands for other pathogens. The consistent results of the inter-batch and intra-batch tests indicated good repeatability of the assay. The detection results of clinical samples and simulated infection samples showed a 100% coincidence rate with the pathogen isolation and identification. [Conclusion] The multiplex PCR assay for the detection of serotypes A, B, and E of Pm was successfully established, which provided technical support for the identification and epidemiological investigation of Pm.