Development of a multiplex PCR assay for detection of different bacterial pathogens associated with reproductive disorders in cattle and buffaloes

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  • PAVITER KAUR Assistant Professor, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India
  • N S SHARMA Controller of examinations, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India
  • A K ARORA Professor, Department of Veterinary Microbiology. Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India


Brucella, Leptospira, Listeria, Multiplex PCR, Mycoplasma


The current study was undertaken to develop a multiplex PCR assay for detection of Brucella, Leptospira, Listeria and Mycoplasma spp. associated with reproductive disorders in cattle and buffaloes. Four pairs of oligonucleotide primers chosen to amplify target DNA regions viz. 31kDa MEM protein in Brucella, 16S rRNA gene in Leptospira, hlyA gene in Listeria and 16S rDNA in Mycoplasma spp. produced amplicon sizes of 223-bp, 331 bp, 456 bp and 270 bp respectively. No amplification was observed when the multiplex PCR was tested against commonly prevalent and related species of bacteria. The sensitivity of the multiplex PCR assay was 116 pg for Brucella, Leptospira, Listeria and Mycoplasma spp. The developed assay was tested for direct detection of the agents in 30 clinical samples of uterine discharges and foetal stomach contents collected from aborted animals and animals with reproductive disorders. By multiplex PCR, out of 30 samples, nine samples of uterine discharges were positive only for Mycoplasma and 1 sample of foetal stomach content was positive for Brucella, Leptospira and Mycoplasma. The multiplex PCR described here appeared to be a rapid and ideal method for detection of all four organisms simultaneously in a single tube reaction and hence can be used for routine diagnostics.


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How to Cite

KAUR, P., SHARMA, N. S., & ARORA, A. K. (2015). Development of a multiplex PCR assay for detection of different bacterial pathogens associated with reproductive disorders in cattle and buffaloes. The Indian Journal of Animal Sciences, 85(12), 1306–1310.