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Evaluation of a Real-time PCR-based assay using the lightcycler system for detection ofToxoplasma gondiibradyzoite genes in blood specimens from patients with toxoplasmic retinochoroiditis

Authors
Journal
International Journal for Parasitology
0020-7519
Publisher
Elsevier
Publication Date
Volume
35
Issue
3
Identifiers
DOI: 10.1016/j.ijpara.2004.11.016
Keywords
  • Toxoplasma Gondii
  • Pcr
  • Real-Time Pcr
  • Toxoplasmic Chorioretinitis
  • Ocular Toxoplasmosis
Disciplines
  • Medicine

Abstract

Abstract PCR based methods have advantages over traditional methods for the diagnosis of toxoplasmosis, especially when serology fails and clinical symptoms are not evident. However, current PCR-based assays are often labour-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. We have developed and evaluated a highly sensitive Real-time PCR (Light-cycler, LC-PCR) to detect and quantify Toxoplasma gondii B1 and bradyzoite specific genes (SAG-4, MAG-1) in serum and peripheral blood mononuclear cells (PBMC) specimens, from five immunocompetent subjects with clinically suspected toxoplasmic retinochoroiditis (TRC) or without a suspected T. gondii infection. A standard curve for quantitation of parasitic load was generated using SYBR Green I fluorescent detection. The results were compared with those obtained with a nested PCR (n-PCR). In TRC patients, both PCR methods confirmed ophtalmoscopy and fluorangiographic findings. Among the TRC patients, the use of LC-PCR was more sensitive than n-PCR for detection and quantification of either B1 gene ( P<0.001) or SAG-4/MAG-1 gene ( P<0.05). LC-PCR has been shown particularly useful to accurately determine the parasite DNA load in follow-up specimens in whom the performance of either B1 or SAG-4 and MAG-1 in detecting T. gondii loads, varied with respect to specific antitoxoplasmic treatment.

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