Abstract
Background/aim Conventional methods of detecting Brucella spp. suffer from technical and biological complications. Besides, newly characterized species of the genus Brucella could be neglected by previously designed polymerase chain reaction (PCR) tests. Therefore, a more accurate PCR-based test seems to be imminently needed.Materials and methods Blood samples were collected from 39 patients diagnosed with brucellosis and 25 healthy controls. Multiple sequence alignments (MSA) were performed on 500 Omp2-related protein and gene sequences. Thereafter, specific primers were designed and synthesized for the regions with highest conservancy. The collected samples were assessed by PCR test. To overcome the cross-reactivity issue, PCR thermal program was optimized regarding annealing time and temperature. ResultsThe MSA results indicated that the N terminus region of the Omp2 protein (DNA 5’ end) is associated with highest conservancy. Primers with highest specificity were designed and synthesized. A two-step PCR reaction was successfully designed and optimized. The desirable bands were observed in clinical samples with high accuracy.ConclusionIt should be pointed out that using a precisely designed primer pair would bring about early infection detection, more success to detect all natural variants and higher cost-to-efficacy ratio in comparison to other detection methods.
Highlights
Brucellosis is a highly contagious zoonosis infection caused by Brucella spp. which are gram-negative, aerobic, nonsporulating, and coccobacillus bacteria
The Multiple sequence alignments (MSA) results indicated that the N terminus region of the Omp2 protein (DNA 5’ end) is associated with highest conservancy
The Omp2 gene is a 1098 nucleotide gene which is translated into a 362 amino acids protein
Summary
Brucellosis is a highly contagious zoonosis infection caused by Brucella spp. which are gram-negative, aerobic, nonsporulating, and coccobacillus bacteria. This infection ( known as Malta fever) is common in human and some animal species [1]. More than 300 million cattle infections (out of the 1.4 billion cattle population worldwide), as well as more than 500,000 annual new human infections, are estimated to be the global disease burden for brucellosis [2]. The members of the genus Brucella are facultative intracytoplasmic parasites traditionally consisting of 6 recognized species. Newly characterized species of the genus Brucella could be neglected by previously designed polymerase chain reaction (PCR) tests. A more accurate PCR-based test seems to be imminently needed
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