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54 Jamali et al.(vaginal discharge, itching, dysuria and dyspareunia) asymptomatic patients, axenically. DNA extractedwere selected and examined for the presence of T. from these isolates was subjected to RAPD analysisvaginalis. From each individual two samples were and amplified with 4 different random primerscollected from the posterior vaginal fornix by using (Fig. 1a-d) show the RAPD patterns obtained with thetwo sterile cotton swabs. First swab was used for wet primers used. All the primers provided distinctmount preparation and the second one was used to patterns. For each primer, the banding pattern wasinoculate the Kupferberg medium (Quelab scored as presence (1) or absence (0) for each isolateLaboratories, Canada). Culture tubes were incubated and matrix table was constructed by SPSS 11.0at 37° C up to 7 days, and examined microscopically software. Dendrogram was built based on RAPD-PCRon days 2, 3, 5 and 7 after inoculation.results for each primer, and for four primers, usingWards method and SPSS 11.0 program (Fig. 2). TheDNA extraction: Log phase T. vaginalis cultures wereisolates with similar banding pattern were assigned aswashed with phosphate buffered saline (pH 7.4) anda single type. OPD1 had the least typing ability as itthe cell pellet was suspended in 400 µl TE (10 mM gave 32 types for typing of 120 strains, whereas OPD5Tris, 1 mM EDTA) buffer (pH 8). To this suspension, 5 had the highest typing ability that gave 58 types. OPD2µl proteinase K (20 mg/ml) and 60 µl of 10% sodium gave 56 and OPD3 gave 43 types. A total of 62dodecyl sulphate solutions were added, and incubated different types were obtained from 120 T. vaginalisovernight at 55-65° C. Following incubation, 100 µl of specimens analyzed. There was one cluster consistingNaCl and 80 µl of pre-warmed (at 65° C)CTAB/NaCl of four patients, seven with three patients, 41 with twosolution were added, vortexed well and incubated at patients and 13 with one patient. According to the tree,65° C for 10 min. DNA was cleaned by adding 700 µl the isolates fell into two major groups (theof chloroform-isoamyl alcohol (24:1) solution and classification results based on discriminant analysisvortoxed for 20 s, and precipitated by 1ml of cold are shown in Table II). The upper branch consisted ofethanol (70%) and centrifugation at 12,000 x g for 5 65 isolates out of which 24 were from symptomaticmin (×2) at 10° C. Finally, after air-drying, the DNA patients and 41 isolates belonged to asymptomaticpellet was dissolved in 50-100 µl TE buffer (pH 8). patients. The lower branch of tree consisted of 55isolates, 50 of them from symptomatic patients andRAPD PCR: Four different 10 base pair primers were only five from asymptomatic ones. The 11 isolates thatused for RAPD analysis (their sequence is shown in were from patients with the history of treatmentTable I; Snipes et al., 2000). The DNA amplification failure, showed a scattered format in the tree (isolateswas performed at final volume of 25 µl containing: 2.5 99, 83, 84, 70, 107, 27, 14, 7, 74, 9 and 36).µl of 10 x PCR reaction buffer (500 mM KCl and 200Table I. The sequence of four primers used for RAPDmM Tris-HCl, pH 8.4), 1.25 µl MgCl 2 (50 mM), 1 µl ofanalysiseach primer (Cinnagen, Iran), 0.5 µl of mixed dNTP(10 mM), 4 µl of template DNA, 15.35 µl of doubleSize (mer) Squence (5' to 3')distilled water and 0.4 µl of Taq DNA polymerase (5OPD 10 ACCgCgAAggunit/µl; Cinnagen, Iran). Negative controls for each of OPD2 10 ggACCCAACCfour primers used contained all components except OPD3 10 gTCgCCgTCAtemplate DNA. The amplification protocol consisted OPD5 10 TgAgCggACAof an initial denaturation step at 94° C for 5 minfollowed by 40 cycle's repetitions of 1 min at 94° C, 1 Table II. Classification result based on discriminantamin at 36° C and 2 min at 72° C. The final cycle “the analysisextension step” was of 15 min at 72° C. The PCRPredicted groupproducts were analyzed by electrophoresis in 1.2%membershipagarose gel in TBE buffer. The gels were then stainedwith ethidium bromide (0.5 µg/ml) and visualizedWard method 1 2 Totalunder the UV transilluminator.Original count 1 62 3 65RESULT2 3 52 55Overall, 4.6% (120/2630) of specimens yielded apositive T. vaginalis culture. Seventy four isolateswere obtained from symptomatic patients and 46 from% 1 95.4 4.6 100.02 5.5 94.5 100.0a. 95% of original grouped cases correctly classified.