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

Parasite. 2007 Jun;14(2):149-54. doi: 10.1051/parasite/2007142149.

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 B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . 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( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.

MeSH terms

  • Animals
  • DNA Probes
  • Fluorescence Resonance Energy Transfer / methods*
  • Gene Amplification
  • Mice
  • Nucleic Acid Hybridization
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Toxoplasma / genetics
  • Toxoplasma / isolation & purification*
  • Toxoplasmosis / diagnosis*

Substances

  • DNA Probes