Establishment and application of a multiplex PCR assay for simultaneously detecting three pathogens inducing abortions in dairy cows

Abstract:

[Background] Brucella, infectious bovine rhinotracheitis virus (IBRV), and Neospora caninum are major pathogens that cause abortions in dairy cows. Current serological methods for detecting the above pathogens are characterized by low specificity and complex operation, while PCR and RT-PCR methods are only capable of detecting single pathogens. Multiplex PCR enabling the detection of multiple pathogens at the same time greatly increases the detection efficiency. [Objective] To establish a multiplex PCR assay for rapidly detecting Brucella, IBRV, and N. caninum in clinical samples. [Methods] Specific primers were designed for Brucella omp25, IBRV gB, and N. caninum SRS2, and the annealing temperature, primer concentration, extension time, and number of cycles of the multiplex PCR assay were optimized. The specificity of the multiplex PCR assay was examined with the standard nucleic acids of Salmonella, Escherichia coli, Cryptosporidium, Toxoplasma gondii, and circovirus. The sensitivity of the multiplex PCR assay was tested with the recombinant plasmids carrying the target genes. Finally, the established assay was employed to test 55 vaginal swabs from cows suffering from abortions. [Results] The optimal conditions of the multiplex PCR assay were annealing temperature of 59 ℃, the extension time of 40 s, 30 cycles, and the primer concentration of 1 μmol/L. The lower limit of detection was 4×10¹ copies for omp25 and SRS2 and 4×10² copies for gB. The established assay showed no cross-reactivity with other pathogens. Nine out of 55 samples were detected positive for Brucella, 3 positive for IBRV, and 7 positive for N. caninum. Particularly, one sample was subjected to mixed infection by Brucella and N. caninum. [Conclusion] A multiplex PCR assay for three pathogens was successfully constructed, enabling rapid and accurate detection of clinical samples.

Key words:dairy cow;abortion;multiplex PCR;Brucella;infectious bovine rhinotracheitis virus; Neospora caninum