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Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR
Reza Mirnejad1, Reza Hosseini Doust, Reza Kachuei
1Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. rmirnejadreza@yahoo.com
Objective:
To evaluate simultaneous detection and differentiates of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method.
Methods:
This study was designed using three primers that could simultaneously identify and differentiate two major species of pathogenic Brucella in humans and animals. Identification and differentiation of each species using the size of the PCR product were determined. To determine the specificity of the method, bacteria close to the genus Brucella were used. Finally, to confirm PCR products, In addition to the products sequence, RFLP was performed on PCR products using restriction enzymes.
Results:
The method of optimized combinatorial PCR in this study could simultaneously detect and differentiate B. abortus and B. melitensis with high specificity and sensitivity in clinical samples. Differentiation of species is based on the resulting bands; therefore, the band 494 bp for B. abortus and 733 bp for B. melitensis were obtained. RFLP and sequencing results confirmed PCR results.
Conclusions:
The results of this study shows that without routine diagnostic methods such as culture and serology tests, using the molecular method of combinatorial PCR, important species of Brucella can be simultaneously identified and differentiated in clinical samples.
