Performance of P30 and B1 PCR

The current PCR method using the P30 gene as target sequence, detected 25-100fg T. gondii DNA, corresponding to the DNA of one tachyzoite (Cornelissen et al. 1984) compared to the 10-100fg obtained with a typical B1 assay (a slight modification of the system described by Burg et al. (1989), performed on the same occasion. These results are in line with the comparison between P30 and B1 PCR made by Wastling et al. (1993). The nested P30 PCR product (266 bp) is more easily discernible on agarose gels than the low molecular sized product (90 bp) of the nested B1 PCR. Also, with the given primers and PCR conditions, use of the P30 system avoids the problems of obscuring smears and extra bands, which have been associated with the B1 method. Although disconcerting, the problems of the B1 system are said to be less disturbing in clinical specimen analysis than in studies of conventional positive standards prepared from infected mice (Mats Olsson, personal communication). Our own laboratory results do not agree here, and this difference in binding performance between the two PCR

systems probably reflects the lower annealing temperature of most published B1 methods (Jones et al. 2000). In one report, the B1 PCR was used for screening purposes whereas P30 PCR was used for confirmation (Hohlfeld et al. 1994). When we analysed suspensions with pre-defined numbers of intact tachyzoites it was found that the P30 PCR, as well as a B1 PCR, was able to produce a positive PCR reaction for one or less than one tachyzoite present in the sample volume processed for T. gondii detection (Figure 29.1). The P30 PCR method as described here, therefore, combines sensitivity with excellent technical performance.

Figure 29.1 Sensitivity of the P30 and B1 toxoplasma PCR systems. Detection of DNA from a known number of parasites inoculated into master mixes as follows: (lane A) master mix only; (B) water; (C) 0.0025 parasites; (D) 0.025 parasites; (E) 0.25 parasites; (F) 2.5 parasites; (G) 25 parasites; (H) 250 parasites per master mix. DNA molecular weight marker, consisting of a mixture of pBR329, cleaved with Bgl I and Hinf I is included. Electrophoresis was run in 1.8% agarose and the gels were stained with ethidium bromide.

Figure 29.1 Sensitivity of the P30 and B1 toxoplasma PCR systems. Detection of DNA from a known number of parasites inoculated into master mixes as follows: (lane A) master mix only; (B) water; (C) 0.0025 parasites; (D) 0.025 parasites; (E) 0.25 parasites; (F) 2.5 parasites; (G) 25 parasites; (H) 250 parasites per master mix. DNA molecular weight marker, consisting of a mixture of pBR329, cleaved with Bgl I and Hinf I is included. Electrophoresis was run in 1.8% agarose and the gels were stained with ethidium bromide.

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