Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Abstract

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3′-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province.

Human Brucellosis Caused by Brucella canis: A Rare Case Report

“…The standard diagnostic method remains to be the isolation of Brucella from blood cultures or host tissues (15). In contrast to other Brucella species, which grow in smooth colonies, B. canis naturally forms rough phase (mucoid) colonies in culture.…”

“…Molecular techniques could be used for diagnosis of human brucellosis. The polymerase chain reaction (PCR) appears to offer several advantages over conventional methods: It is easy to perform, rapid, and safe for laboratory staff because serum-based PCR-assay will reduce the risk of handling the microorganism (6,15). In this study, specific PCR is used for diagnosis and confirmation of B. canis, which was reported by biochemical and culture methods.…”

Diagnosis of Caprine Brucellosis by Serology and Multiple PCR

“…In the present investigation, 30 serum samples were confirmed as positive for RBPT, and from the same sampling with 30 samples of whole blood, the isolation was confirmed of B. melitensis with multiple PCR. In the same manner, Irajian et al analyzed 68 isolations by PCR from humans as well as from animals, among which B. melitensis predominated in 36 isolates, two of B. abortus and one of B. suis of the animal specimen, and 24 isolates of B. melitensis and six of B. abortus of the human specimen [8]. In this regard, it was documented that B. melitensis presents very severe clinical conditions in humans [2].…”

“…We added, to the cellular extract, 30 µL of Sodium DiSulfate (SDS) at 20% and incubated this at 37°C during 5 min. We extracted the lysate with a similar volume of phenol-chloroform-isoamyl alcohol (24:25:1) u/u; this was mixed and centrifuged at 13,000 rpm for 5 min (206.4:215:8.67 µL) [8]. The aqueous phase was transferred, we added 30 µL of sodium acetate 3M, and filled the tube with cold ethanol at 95%.…”

“…Extraction of genomic DNA in whole blood consisted of placing 500 µl of whole blood in an Eppendorf tube, to which we added 1 mL of red-blood lysis solution, 0.01 M Tris-HCl pH 7.6, 320 mM sucrose, 5 mM MgCl 2 , and 1% Triton X 100 8 . This was centrifuged at 7,000 rpm for 2 min, the supernatant was discarded, and this step was repeated two or three times until we observed no excess of hemoglobin.…”