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OJVRTM

Online Journal of Veterinary Research©

Volume 13 (1):76-85, 2009

Evaluation of an Elisa-PCR detection method for Brucella spp.

Nima K¹ , Reza HD², Ashraf MM¹, Bahman T³.

1, Department of Bacteriology, Tarbiat Modares University, Tehran/Iran, 2,Department of Microbiology / Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences. P.O.Box: 14145-413, 3, Iran Institute Pasteur

ABSTRACT

Nima K, Reza HD, Ashraf MM, Bahman T, Evaluation of an Elisa-PCR detection method for Brucella spp. Online J Vet Res, 13(1):76-85, 2009 Brucellosis is a worldwide re-emerging zoonosis causing high economic losses and severe human disease. Consequently, rapid and reliable, sensitive and specific, easy to perform and automated detection systems for Brucella spp. are urgently needed to allow early diagnosis and adequate antibiotic therapy in time. In attempt to improve current molecular detection of Brucella species, we developed a ELISA - PCR, using our own designed primer set and probe. A new primer sets based on "omp-31" sequence of B. melitensis 16M were designed. Its specificity was checked with appropriate online bioinformatics softwares. The primer specificity was confirmed by testing the reaction with non-Brucella strains. A biotinylated probe complementary to an internal sequence of the PCR products was designed. The labeled non-specific fragments bound to streptavidin-coated wells, saturating the solid phase streptavidin by biotin-streptavidin interaction. The new protocol was able to detect 5 fg of Brucella genome after appropriate optimization. Few human serum, whole blood and also different affected tissue samples from slaughtered livestock with brucellosis were used for protocol evaluation. Further samples should be tested before final conclusion about the results.

Keywords: Brucellosis, B. melitensis, B. abortus, omp-31, ELISA-PCR, Digoxigenin

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