Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control.
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.
Brenier-Pinchart
M P
MP
Parasitologie-Mycologie, Département des Agents Infectieux, Centre Hospitalier Universitaire, BP 217, 38 043 Grenoble Cedex 9, France. MPBrenierPinchart@chu-grenoble.fr
Morand-Bui
V
V
Fricker-Hidalgo
H
H
Equy
V
V
Marlu
R
R
Pelloux
H
H
D000818:Animals; D015342:DNA Probes; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005784:Gene Amplification; D051379:Mice; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis*
D015342:DNA Probes