Amplification procedures

Presence of T. gondii DNA was detected in a nested PCR system, where the outer primers 5'-CACACGGTTGTATGTCGGTTTCGCT-3' (forward, nt 149-172; Gene Bank Accession Number S63900) and 5/-TCAAGGAGCTCAATGTTACAGCCT-3/ (reverse, nt 520-497), were designed by Weiss et al. (1991), from the published P30 sequence (Burg et al. 1988). This system gave an outer product of 372 base pairs. The inner nesting primers were 5/-TGACGAGTATGTTTCCGAAGGC-3/ (corresponding to nt 203-224) and 5/-TGGGCAGATTTGCCTGTTGGGT-3/ (nt 468-447), amplifying a region of 266 base pairs. The specificity of the method was ascertained: (i) by using material from autopsies and PMBC of T. gondii-infected and uninfected mice, as described (Weiss et al. 1991); (ii) by cross-checking with our standard DNA positive controls for CMV, HSV, EBV, and VZV PCR (reviewed by Bergstrom et al. 1995); and (iii) by oligonucleotide hybridization of the PCR products using the 5'-biotin labelled probe (5/-biotin-CTCACACCGACGGAGAACCACTTC3/) (nt 370-393) described by Weiss et al. (1991).

Master mixes were prepared using the thermostable DNA polymerase (AmpliTaq, Applied Biosystems) at a concentration of one unit TAQ per reaction mix of 50^l, 10mM Tris HCl (pH 8.3 at 20°C), 1.5 mM MgCl2, 50 mM KCl, 200 •M each dNTP, and with 0.2^M of the forward and reverse primers in the outer or inner PCR reaction mixes.

Sample DNA was added to the reaction mixes as 5, 8, or 10^1 of H^O-solubilized, concentrated DNA. In the case of placenta, biopsies, and brain tissue, DNA preparations were also tested in dilution to reduce problems of inhibition or DNA overload. To detect and avoid the problems of cross-contamination between specimens, water controls were set between each sample preparation and precautions taken as described by Kwok and Higuchi (1989). Target DNA was amplified in the 9600 thermocycler (Applied Biosystems) with an initial cycle programme of 30 cycles followed by a nesting round of 40 cycles. In the first

PCR, a denaturation step of 94°C for 2min 30s was followed by a 30 cycle programme of denaturation at 94°C for 15 s, primer annealing at 65°C for 10s, and primer elongation at 72°C for 15 s, with a time extension at 72°C of 1s per cycle. Five microlitres of the first-round PCR were 'nested' into the inner primer system mixes and subjected to similar cycling conditions, with the exception that amplification was continued for 40 cycles. PCR products were detected by gel electrophoresis of 15^l of reaction mix on a 1.8% agarose with ethidium bromide (0.2^g/ml) giving fluorescent staining of nucleic acid amplicons on exposure to UV light.

To define the problem of false negative PCR reactions caused by inhibitors present in the sample DNA, for example, iron from haemoglobin, repeat PCR reactions were performed using ß-globin primers and specimen DNA. 'Spiking' of repeat toxoplasma primer PCR mixes with the specimen DNA sample plus control T. gondii positive DNA was also used to detect false negative reactions caused by sample inhibition.

Toxoplasma positive samples were confirmed by hybridization of the amplified products with a specific biotin labelled probe, indicated above (Weiss et al. 1991). In some instances, the PCR products were sequenced, using the ABI FS dd NTP D-rhodamine cycle sequencing kit, and the labelled products analysed on the ABI 310 (Applied Biosystems).

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