Comparison of duplex polymerase chain reaction and Rose Bengal test for diagnosis of brucella abortus and brucella melitensis

BACKGROUND: Diagnosis of brucellosis requires a rapid and accurate method such as the polymerase chain reaction (PCR). The purpose of this study was simultaneous detection of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) in serum samples using the duplex PCR technique and then comparing the results using the Rose Bengal test (RBT). METHODS: In this comparative-descriptive study, 100 serum samples were collected from a veterinary station in Shahriar City, Iran. Moreover, the monoplex-PCR of B. abortus and B. melitensis and duplex-PCR for both agents was optimized. The limit of detection (LOD) and specificity test were also checked. Besides, deoxyribonucleic acids (DNAs) were extracted from the serum samples by the DNA extraction solution (DNG-plus) technique. The PCR product was cloned in pTZ57R plasmid by T/A cloning. RESULTS: B. abortus (494bp) and B. melitensis (733bp) amplicons were observed in 1.5% gel electrophoresis. The LOD of the monoplex-PCR test for both of the agents was 100 genomes per reaction. Additionally, 40 out of 100 samples were positive for RBT, out of them, 35 samples were positive with duplex-PCR, 31 samples were positive for B. abortus, and 4 for B. melitensis; moreover, 20 samples were positive with duplex PCR from 60 negative RBT. This number detected 17 samples of B. abortus and 3 samples of B.melitensis. CONCLUSION: The number of positive samples by duplex-PCR was more than the RBT; therefore, we can assert duplex-PCR for confirming the RBT results.