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Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control
Authors:Brenier-Pinchart M P  Morand-Bui V  Fricker-Hidalgo H  Equy V  Marlu R  Pelloux H
Institution:Parasitologie-Mycologie, Département des Agents Infectieux, Centre Hospitalier Universitaire, BP 217, 38 043 Grenoble Cedex 9, France. MPBrenierPinchart@chu-grenoble.fr
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.
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