Dr. Murai Éva - Gubányi András szerk.: Parasitologia Hungarica 31. (Budapest, 1998)
Fig. 1. Detection of Toxoplasma gondii Bl gene by first stage PCR. The ethidium bromide stained gel is shown. Lanes 1 and 10, pUC19-fispR 1 molecular weight standards lane 2, positive control (tachyzoites from mouse intraperitoneal aspirate) lanes 3 to 8, different urine samples lane 9, negative control (reagent control) Serology: Sera from newborns and their mothers were tested by the complement fixation test (CFT) using the Toxoplasma antigen of the SEVAC firm (SEVAC Ltd., Prague, Czech Republic) (Szénási et al. 1996-1997; Szénási et al. 1997a; Szénási et al. 1997b). Specimens were further examined with commercially available anti-P30 IgM and IgA immunocapture ELISA kits (Sanofi Diagnostics Pasteur, S. A., Marnes-LaCoquette, France), using the P30 protein as antigen. The protocol for the ELISA detection has been described by the manufacturer. Processing of urine samples: Twenty to 30 ml of urine was collected from the neonates. Urine samples (5-10 ml) were centrifuged at 1800 g for 10 minutes and the pellets were washed 3 times in physiological saline solution and stored at -20 ° C until examination. The pellets were incubated in lOOul of lysis buffer (10 mM Tris HCl pH 8.3,1.5 mM MgCb, 50 mM KCl, 0.1 mg/ml gelatin, 0.5 % Tween 20, and 20 ug of proteinase K Sigma Chemical Co., St. Louis, MO, USA) at 55 0 C for 90 minutes (Nicoll et al. 1996, Fuentes et al. 1996) for DNA extraction. Inactivation of proteinase K was carried out at 94 0 C for 10 minutes and followed by centrifugation of the suspension at 12,000 rpm for a few seconds. Polymerase chain reaction: The 1234 5 6 7 8 9 10 first stage DNA amplification was carried out in a PCR mixture of a reaction volume of 100 ul containing 10 mM Tris HCl pH 8.3, 0.2 M each (Pl and P2) primers, 50 mM KCl, 1.5 mM MgCb, 0.01 % gelatin, 2 mM dNTP mix (clATP, dGTR dCTP and dTTP), 2.5 IU Taq polymerase (WestTeam Biotech Ltd., Budapest, Hungary) complemented with lysed samples (Fuentes et al. 1996). The target DNA in the samples to be detected by PCR is a part of the sequence of T. gondii 35-fold repetitive Bl gene (Burg et al. 1989, Fuentes et al. 1996). The sequences of the primers used were: Pl: 5-GGAACTGCATCCGTTCATGAG-3' (694-714 nucleotides), P2: 5'-TCTTTAAAGCGTTCGTGGTC-3 ' (887-868 nucleotides). The amplification protocol consisted of an initial denaturation: 90 0 C for 10 minutes (1 cycle), a 3-temperature PCR: 94 0 C for 1 minute, 55 0 C for 1 minute, 72 ° C for 1 minute (31 cycles) and a terminal extension: 72 0 C for 10 minutes (1 cycle). The amplification procedures were carried out in a Perkin Elmer GeneAmp PCR System Fig. 2. Detection of Toxoplasma gondii Bl gene by nested-PCR. The ethidium bromide stained gel is shown. Lanes 1 and 10, pUC19-ÄspR 1 molecular weight standards lane 2, positive control (tachyzoites from mouse intraperitoneal aspirate) lanes 3 to 8, different urine samples lane 9, negative control (reagent control)
