Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 53 issue 6 (june 2019) : 821-826

Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp

Aparajita Das, Bablu Kumar, Soumendu Chakravarti, Karam Pal Singh, Abhishek, Garima Shrinet
1Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243 122, India.
Cite article:- Das Aparajita, Bablu Kumar, Chakravarti Soumendu, Singh Pal Karam, Abhishek, Shrinet Garima (2018). Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp. Indian Journal of Animal Research. 53(6): 821-826. doi: 10.18805/ijar.B-3584.

ABSTRACT

A simple single-tube duplex-PCR assay was optimized for rapid and sensitive detection and differentiation of Brucella abortus (B.abortus) S19 vaccine strain from other Brucella spp.(B.abortus 544,B abortus S99,B.melitensis 16M,B.suis).This assay was optimized using two primer pairs that were designed, one targeting genus specific multicopy IS711 and another eryC for the development of the duplex-PCR assay. Specificity of the assay was assessed using DNA templates from various Brucella species (B.abortus, B.melitensis, B.suis) and non Brucella bacteria like Staphylococcus aureus, Proteus vulgaris, Pasteurella multocida, Pseudomonas aeruginosa, Citrobacter freundii, E. coli, Salmonella enteritidis, Campylobacter jejuni and Listeria monocytogenes. The assay was also evaluated on spiked milk samples with known B.abortus cultures. The assay was able to detect up to 2.47 x 103CFU of B. abortus S99 organism in spiked milk sample. The assay was further validated in 53 clinical specimens (aborted foetal stomach content). Results confirm that Single tube duplex-PCR assay was useful for early and rapid detection and differentiation of B. abortus vaccine strain S19 from other Brucella spp in milk as well as in clinical specimen. 

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