Quantitation of Toxoplasma gondii DNA in a competitive nested polymerase chain reaction

Aim-To quantify Toxoplasma gondii DNA using a specially constructed artific ial template as competitor in a nested polymerase chain reaction (PCR). Methods-The diagnostic assay was a nested PCR employing four primers that a mplify part of the single copy gene for the P30 major surface antigen in T gondii. An artificial competitor containing the four primer binding sites w as made first by creating a 216 bp deletion in the native 914 bp full lengt h PCR product using restriction enzyme digestion, ligation of selected dige stion fragments, and cloning the ligation product into an E coli plasmid ve ctor for production. Competitive nested PCR using three different quantitie s of T gondii genomic DNA with four corresponding 10-fold dilutions of the artificial competitor was then performed, and the results visualised with a garose gel electrophoresis. A standard curve was drawn by plotting the T go ndii to competitor ratio readings against log,, of the competitor readings. Results-The band intensities on agarose gel showed quantitative amplificati on in competitive nested PCR. The amount of competitor required to achieve equal molar amounts of PCR products is calculated by reading off the value of the competitor where the T gondii to competitor ratio equals 1 on the st andard curves. Conclusions-Competitive PCR is possible with a nested assay, and quantitati ve amplification is well preserved. The use of an artificial competitor con taining the same primer binding sites as the target enables the absolute am ount of T gondii DNA in unknown samples to be estimated. In addition, the c ompetitor simultaneously serves as a control for detecting false negative r esults of failed reactions in individual assay runs.