Sensitive and specific detection of T. gondii DNA is afforded by
nested PCR (nPCR). For this procedure, two pairs of PCR primers
are designed to flank a polymorphic locus. The first pair of
external primers is designed to amplify a region surrounding the SNP of
interest. A second pair of primers (nested primers) are designed
within the amplicon generated by the first pair. A second round of
amplification increases sensitivity and specificity. Following the
second round of amplification, the DNA is digested with a diagnostic
restriction enzyme that is chosen to detect a SNP between different
strains of the parasite. This approach greatly improves the
utility of PCR for typing samples obtained from clinical samples, where
DNA content of the parasite is generally limited.
We have previously used nPCR to type strains of T. gondii
isolated from AIDS patients based on the polymorphic SAG2 locus
(Howe et al. 1997).
However, the limitation of
using a single locus is that mixed strains or exotic genotypes will be
misclassified as having a simple genotype. Consequently, we have
developed nPCR markers for four unlinked loci. When used in
combination, these markers provide sensitive typing for the major
lineages of T. gondii. They should also provide greater
sensitivity for identifying mixed or exotic genotypes.
The markers we have developed for nPCR as are based on the following
(choose marker to open a pdf file that provides complete information).
We have tested these on human blood samples consisting of buffy coats
that were purified using a QIAamp blood kit (Qiagen Inc., Chatsworth,
Calif.). Each marker is capable of detecting as few as five
parasites in the presence of host DNA. Analysis of additional
clinical samples will provide a better understanding of the association
between parasite genotypes and human toxoplasmosis. Interested
parties are encouraged to contact us if they would like more specific
information on the methodology or if they have samples they would like