AL-ATTAS ET AL
concentration and purity of the DNA were determined spectrophotometrically by reading A260 and A280. DNA Amplification
The primers B4 (5-TGGCTCGGTTGCCAATATCAA
3) and B5 (5 CGCGCTTGCCTTTCAGGTCTG-3),
described previously by Bailey et al.,7 were used to amplify a target sequence of 223-bp within a gene code for the production of a 31-kDa membrane protein specific to the genus Brucella.8 PCR was performed using a protocol described elsewhere.4 Briefly, a 50 µL volume reaction mixture containing 10 mM tris-HCl (pH 8.4), 50 mM KCl, 1 mM MgCl2, 200 µM each deoxyribonucleotide triphosphate (dATP, dGTP, dTTP, dCTP, Pharmacia LKB
polymerase (Perkin-Elmer Cetus Co., Norwalk Conn., USA), oligonucleotide B4 and B5 (100 nM each; Gulf Biotech, Riyadh, Saudi Arabia) and 2-4 µg of total DNA extracted or 150 ng from the positive control was processed in a thermocycler (Perkin-Elmer 9600). The cycling conditions consisted of initial denaturation at 93°C for 5 min., followed by 35 cycles of 60 sec. of template denaturation at 90°C, 30s of primer annealing at 60°C, and 60 sec. of primer extension at 72°C and final extension at 72°C for 7 min. Specificity of the assay was examined by testing E. coli (ATCC No. 25922) DNA as targets. DNA free control was also included to monitor contamination.
Each sample was tested at least in duplicate. Generally recommended procedures were used to avoid contamination. The products (10-18 µg from each reaction mixture) were analyzed by agarose gel (1.5%) electrophoresis at 160 v for 15 min., stained with ethidium bromide and photographed on a UV Tran illuminator. The presence of a clear-cut band was considered as a positive result. The sensitivity of the assay was tested by decreasing the amount of target DNA in the reaction mixture through serial dilutions of total DNA of one positive case and processing, as described above.
DNA Extraction from Culture
Positive control with genomic DNA isolated from a suspension of Brucella spp. was extracted using a modified alkaline lysis method described by Bernboim and Doly (1979) and Ish-Horowicz and Burke 1981.9 Briefly, colonies of Brucella spp. were harvested from chocolate agar, killed in 67% methanol, and pelleted by centrifugation at 15,000 Xg for 3 min. after addition of distilled water. The supernatant was drained away completely to keep the pellet dry as much as possible. The pellet was then resuspended in 100 µL of solution I (50 mM glucose, 25 mM tris-Cl [pH 8], 10 mM EDTA), followed by the addition of 200 µL of solution II (0.2 N NaOH, 1% SDS), mixing and storage on ice. Finally 150 µL of solution III (5 MK acetate 60 mL, glacial acetic acid 11.5 mL, H20 28.5 mL) was added, tubes were mixed thoroughly and centrifuged at 15,000 Xg for 5 min. at 4°C,
Annals of Saudi Medicine, Vol 20, Nos 3-4, 2000
the supernatant was discarded and the DNA extracted as described above.
Of the 17 blood samples obtained from patients with brucellosis, eight were positive by culture (Table 1), with a mean detection time of 7 days (range 4-14). All the samples showed high-titer antibrucella antibodies, including the three samples submitted after treatment. PCR was positive only in the 14 samples which were collected before treatment during active infection and the one case of relapse, thus representing 100% sensitivity. PCR was negative in the remaining three samples that were submitted after treatment. Samples containing E. coli DNA and DNA free target were negative in all assays. Among the 33 controls, 29 had negative results by PCR (Table 2), and the four false-positive cases were all from the occupationally exposed group. These were asymptomatic herdsmen, one with a history of febrile illness and treatment two years previously. None had a positive blood culture or detectable antibrucella antibodies, thus accounting for 88% specificity. The dilution method showed that as little as 30 fg of total DNA can give a visible band.
The high endemicity of brucellosis in the Arabian Peninsula has stimulated many researchers to research many of its aspects, such as epidemiology and clinical features. However, there have only been a few reports covering the diagnostic issues. This has led others10 to call for expanding research, particularily on the use of new technology to address unsettled diagnostic issues relating to brucellosis. (We report the results of PCR as compared to serology and culture methods for the diagnosis of brucellosis). The PCR is currently used for the diagnosis of many infectious diseases. The publication of Queipo- Ortuno et al.4 provided the basis for this study. Our PCR- based assay was able to identify all 14 samples submitted during active infection, which gave a sensitivity of 100%. This high sensitivity is probably related to the ability of the PCR-based assay to detect as little as 30 fg of total DNA. This dilution method also showed that the assay could be used with a wide range of target concentration. Romero et al.11 found that the lowest detection limit using DNA from culture isolates is 80 fg. Despite the different source of DNA that we used, our assay was sensitive enough to detect Brucella genome from 30 fg of total DNA. The three samples which were submitted after completing treatment were negative by PCR, indicating the potential