Comparison between conventional PCR and PCR - ELISA for detection of Brucella melitensis

Abstract

Molecular detection techniques are believed to be key tools for both prevention and treatment follow up of brucellosis within live stock and human beings. Consequently rapid, reliable, easy to perform and automated systems for Brucella detection are urgently needed to allow early diagnosis and adequate antibiotic therapy in time. Brucellosis is a worldwide re-emerging zoonosis causing high economic losses and severe human disease. In attempt to improve current molecular detection of Brucella melitensis, we compared a conventional PCR with PCR- ELISA, to detect brucella genome within standard strains and clinical isolates. Primer sets based on “omp-31" sequence of B. melitensis 16M were designed. The primer specificity was checked with appropriate online bioinformatics softwares. The primer specificity was also 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. Compared with conventional PCR, the PCR- ELISA proved to have more sensitivity for 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.