NESTED PCR Analysis

 

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 loci: SAG2-5SAG2-3, BTUB, GRA6 and SAG3 (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 analyzed.

 

 

ToxoDB

ApiDots

ToxoGMD


Sibley Lab (sibley@borcim.wustl.edu)
Department of Molecular Microbiology
Washington University School of Medicine
St. Louis, MO USA

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