Technical background

One major problem associated with the laboratory diagnosis of T. gondii infections is that a clinical specimen containing only a few tachyzoites can reflect a life-threatening infection.

Thus, PCR-based methods for laboratory diagnosis of T. gondii infection should be able to detect literally single copies of the genome in such specimens, and several PCR systems with adequate sensitivity have been published (Burg et al. 1989; Weiss et al. 1991). To accomplish this, at least three genes of the T. gondii genome have been evaluated as target sequences for PCR, namely the B1, P30, and 18S rDNA genes (Jones et al. 2000).

Most systems have acceptable sensitivity and specificity, and one example of a successful PCR method is that based on the gene encoding the P30 surface membrane antigen (Savva et al. 1990; Weiss et al.

1991). The probability of detection of T. gondii should be increased if regions of DNA containing repeat sequences are used. As such, the B1-gene, containing 35 tandem repeats (Burg et al. 1989; Ho et al.

1992), and the ribosomal RNA-gene (Cazenave et al. 1992) with 110 repeats (Guay et al. 1993), offer a theoretical limit of 0.01 tachyzoite of T. gondii per sample assay (Macpherson and Gajadhar 1993). These detection systems do provide very high potential sensitivity. However as discussed below, there are some advantages in the P30 PCR method compared with the methods employing repeat DNA sequences as target, despite the inherent high theoretical sensitivity of the latter.

Developments of the PCR-based diagnosis of T. gondii infections now include scoring of the pathogenic properties of different T. gondii strains (Guo et al. 1997). One approach to identifying T. gondii strains associated with human pathogenicity was based on short term cell culture propagation and subsequent PCR, using the surface antigen SAG2 locus (Howe et al. 1997). Rapid identification and discrimination of T. gondii types have been achieved by multilocus PCR-RFLP of SAG3, and SAG4 (single copy surface antigen genes) as well as SAG1, SAG2, and B1 genes (Grigg et al. 2001a,b). Although chronic infections in humans and animals are said to be associated predominantly with the mouse avirulent types II or III, correlation between clinical disease, pathogenicity, and genotype in human infections is still under study (Honore et al. 2000; Fuentes et al. 2001; Grigg et al. 2001b). Genotyping with PCR-RFLP methods may provide new approaches for investigation and diagnosis of T. gondii infections, but the significance of their contribution to the laboratory diagnosis remains to be determined. Here, we present a PCR system using the P30 gene as the target sequence, which according to our experience combines good sensitivity, 25-100fg of T. gondii DNA, corresponding to one or less tachyzoite, and high specificity.

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