Molecular Survey of Brucella melitensis Field Isolates using Sequence-Based PCR of Outer Membrane Protein ۳۱

Sequence-based Polymerase Chain Reaction (PCR) has been introduced as an effective and reliable method for bacterial strain typing, which could provide a reliable typing approach for clinical laboratories. This study aimed to describe the reproducibility and performance of the Outer Membrane Protein ۳۱ (Omp۳۱)-based PCR, as a molecular genotyping tool for Brucella melitensis (B. melitensis) typing. The ۳۱ KD outer-membrane protein of Brucella, which encodes the Omp۳۱ gene, can be applied as an antigen to diagnose brucellosis. For this purpose, ۱۴۶ samples were taken from human blood samples, bovine and camel lymph nodes, as well as sheep and goat aborted fetuses, including fetal kidney, abomasum, liver, lung, spleen, and heart for bacteriological investigation. The molecular detection of the Omp۳۱ and IS۷۱۱ genes was performed using the isolated B. melitensis (n=۱۴). The sequencing of the Omp۳۱ gene of B. melitensis in the Iranian field isolates was also performed for the whole gene sequencing. The homology of all sequences was then checked with the reported National Center for Biotechnology Information sequences using a basic local alignment search tool for the nucleotide diversity evaluation.  The findings revealed that B. melitensis isolates were recovered from ۱۴ examined cases and confirmed by the IS۷۱۱-based PCR with a PCR product of ۷۳۱ bp. Moreover, ۱۴ Iranian B. melitensis sequences clustered together as a monophyletic grouping with bootstrap support of ۶۳, and they were closely related to the B. melitensis reference isolates. This Omp۳۱-based phylogenetic placement strongly indicates the monophyletic origin of the Iranian B. melitensis in different animals and human hosts.

Keywords:

Brucella melitensis , Omp۳۱ , Phylogenetic analysis , Sequence-based PCR