Area and time of study
The patient samples were gathered from different provinces in Iran, during a period of 10 months (from October 2017 to July 2018). Two patients from Babol with geographical specification of latitude: 38° 35´N and longitude: 44° 58´E at 211 km northeast of Tehran , 2 from Shiraz with latitude: 29° 59´N and longitude: 52° 58´E at 922 km south of Tehran , 6 patients from Mashhad with latitude: 36° 20´N and longitude: 59° 35´E at 898 km east of Tehran, 4 from Borujerd with latitude: 33° 55´N and longitude: 48° 50´E at 386 km southeast of Tehran, 4 from Urmia with latitude: 37° 54´N and longitude: 45° 07´E at 761km northwest of Tehran, 3 from Makoo with latitude: 39° 15´N and longitude: 44° 31´E at 873 km northwest of Tehran,24 from Khoy with latitude: 44° 58´N and longitude: 38° 33´E at 782 km northwest of Tehran, and 4 from Tabriz with latitude: 38° 09´N and longitude: 46° 27´E at 618 km northwest of Tehran.
Case definition and data collection
Blood specimens were collected from 49 suspected cases of patients with brucellosis symptoms who were referred to diagnostic laboratories in different cities from northern (2 cases, 4.08%), southern (2 cases, 4.08%), western (39 cases, 79.59%) and eastern (6, 12.24%) provinces in Iran, during a period of 10 months. Serum samples were processed on the same day as blood collection. Before blood collection, the written informed consent was obtained and the questionnaire which included age, genus, job, residence area, primary clinical symptoms was filled for each patient.
Prior to amplification, the isolated specimens (sera) were tested by the serological assay of 2ME (2-Mercaptoethanol) test. Then, in accordance with standard methods, a positive 2ME titer was defined as either equal or greater than 1:80 and Coombs Wright titer was considered as either equal or greater than 1:80 . In this study, we gathered samples from suspected patients which their titer of the 2ME test was equal or higher than 1/20 and serial dilutions of serum samples were prepared as follows: 1/2, 1/10, 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280. The serological tests were checked by positive and negative controls .
Bacterial DNA extraction
The viable bacterial samples of B. melitensis and B. abortus were provided from the microbial culture collection of Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran. The DNA templates belonging to the aforementioned bacteria were extracted by the commercial kit (GTP. Tehran, Iran). The purified genomic DNA was stored at -20° C until the day of amplification, followed by a serial dilution of purified DNA and the DNA of Escherichia coli was extracted and used as the negative control.
Extraction of DNA from serum samples
The isolated patient serum samples were kept in tubes containing sodium citrate. The DNA was extracted from the serum specimens of 200 µL volume, according to the guideline of the kit.
Bioinformatics analyses, primer design and DNA amplification
Two primer sets with different target genes were used after bioinformatics analysis:
First, B4 (5´-TGG CTC GGT TGC CAA TAT CAA-3´) and B5 (5´-CGC GCT TGC CTT TCA GGT CTG-3´), with a target gene encoding a 31-kDa B. abortus antigen which is a conserved sequence in all species of Brucella . The reaction was consisted of 12.5 µL 2X PCR master mix (Amplicon, Denmark), 5 µL DNA template, 0.5 µL of each primer and nuclease-free water up to 25 µL. The amplification was done with Techne, touch gene gradient PCR machine, model: Techne TC-512 (WWW.Techne.com). The thermo-cycler was programmed as follows: Initial denaturation at 95°C for 5 min, 35 cycles of template denaturation at 94°C for 1 min, 30 S for primer annealing at 60°C and 60 S for primer extension at 72°C with final extension cycle at 72°C for 7 min.
Second, IS711 specific primer which was designed based on the sequence of B. melitensis deposited in the GenBank. The primer was designed applying Codoncode Aligner software (V.7.1.2)). The designed primers, F (5´-CGC TCG CTG CCA TAC TTG CA-3´) and R (5´-CTG AAC AAG CCG GGC CTG AT-3´) amplified a 448 bp fragment which was a repetitive genetic element of IS711 and was unique to Brucella species. At least, one copy of this repetitive genetic element may appear as a common locus in all species of Brucella . The IS711 PCR assay was carried out in total volume of 25 µL containing the same mixture which was used for PCR. The gene amplification using the IS711 primer was programmed as follows: initial denaturation at 95°C for 5 min. 35 cycles of template denaturation at 94°C for 1 min, 60 S for primer annealing at 63°C and 60 S for primer extension at 72°C with final extension cycle at 72°C for 7 min.
In each PCR assay, a positive control, extracted DNA from B. melitensis Rev. 1 and B.abortus S19 and negative control, extracted DNA from E. coli (ATCC 35218) were applied to control the running process and the absence of cross-contamination. All the standard items were checked for prevention of any probable contamination . The tests were carried out twice. After the amplification process, the samples were run on 1% agarose gel (Sigma). The gel was stained by1 µg/ml ethidium bromide and after destaining, the DNA bands were visualized within Gel documentation UV chamber.
In the current study, for colony forming unit (CFU) estimation, a 48h incubated suspension of B. melitensis and B. abortus within sterile PBS was used for preparing serial dilutions from 10-1 to 10-10. From each dilution, 0.1 ml was streaked onto the Brucella agar and was incubated at 37°C for 72h. Then, the colonies of B. melitensis and B. abortus were counted  and the bacterial concentration was calculated to be about 5 ×108 CFU/ml for both B. melitensis and B. abortus. Then a serial dilution of extracted purified DNA of B. melitensis and B. abortus was prepared from 10-1 to 10-10. Afterwards, five microliters of each dilution was used as template in the PCR process. No amplification was detected with E. coli DNA template. The tests were carried out twice.