Design and optimization of single reaction Multiplex Nested PCR for enrichment of PCR product in detection of Brucella

Bacteria of the Brucella are responsible for zoonotic infections. Detection of pathogenic species in different country is important. In this research three type sample, pure isolate, Milk and serum were analyzed by molecular approach. We examined peripheral blood from 30 patients affected by brucellosis, and 100 milk random samples collected from ethnic ranches. Two PCR products was sequenced to confirm identity of amplicons. DNA extraction from all of samples was performed by using a SDS/NET/Proteinase-k lyses procedure followed by phenol-chloroform standard purification method. Presence of Brucella melitensis was identified in 22 cases (73.3%) of patients that were confirmed by serological means and in 14 of milk samples(14%). However it must be considered positive results in vaccinated animals. We optimized a very sensitive reaction, by using of specific primers for amplification of a region of omp31 gene in different species of Brucella in the first round of PCR reaction that followed by a second reaction with very specific nested primers for Brucella melitensis omp31 gene. Primers designed as two PCR products in the first and second round of reaction had a near size (136 and 128bp respectively) and enrich one another. As a result, their bounds in Agarose gel electrophoresis were stopped in the nearly same location. we developed a simple method with high sensitivity, and it may therefore be considered a useful tool for diagnosis of human brucellosis.