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OJVRTM

Online Journal of Veterinary Research©

 

Volume 13 (1):76-85, 2009.  Editors extensive redaction 2018.

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

 

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

 

1Department of Bacteriology, Tarbiat Modares University, Tehran, 2Department of Microbiology / Research Center of Molecular Biology, Baqiyatallah University of Medical Sciences, 3Iran Institute Pasteur, Iran.  

 

ABSTRACT

 

Nima K, Reza HD, Ashraf MM, Bahman T, Evaluation of an Elisa-PCR detection method for Brucella spp. Onl J Vet Res, 13(1):76-85, 2009.  To improve detection limit of conventional PCR for Brucella spp, we designed a new primer set optimized and hybridized on microtiter plate enzyme immunoassay. We report an ELISA – PCR for detection of Brucella spp. Primer sets based on "omp-31" sequence of B. melitensis 16M specificity confirmed by reaction with non-Brucella strains. A biotinylated probe complementary to an internal sequence of the PCR products was designed wherein labeled non-specific fragments were bound to streptavidin-coated wells, saturating the solid phase streptavidin. The protocol was able to detect 5 fg of Brucella genome after optimization. Human serum, whole blood and tissues from slaughtered livestock with brucellosis were used for protocol evaluation. Our results confirm high specificity over a wide panel of microorganisms. The method is rapid, easy requiring no electrophoresis apparatus, UV light, or darkroom, or use of toxic chemicals such as ethidium bromide. The technique allows simultaneous handling of a large number of samples and can be automated. The Elisa–PCR may be useful for diagnosis of brucellosis both in humans and livestock.   

 

 

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

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