Adapting a conventional pcr assay for Toxoplasma gondii detection to real-time quantitative pcr including a competitive internal control

Abstract

We have developed a quantitative PCR assay (LightCycler®) using the pair of primers JW58 and JW59 for the detection of the 35- fold repeated B1 gene of Toxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of T. gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106 parasites in one ml of amniotic fluid (1 to 105 T. gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.

Keywords

• Toxoplasma gondii
• LightCycler®
• real-time PCR
• B1 gene
• internal amplification control