1. Design of a one-tube hemi-nested PCR for detection of Toxoplasma gondii and comparison of three DNA purification methods.
- Author
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Pujol-Riqué M, Derouin F, Garcia-Quintanilla A, Valls ME, Miró JM, and Jiménez De Anta MT
- Subjects
- Animals, DNA Primers, DNA, Protozoan analysis, Gene Amplification, Humans, Reagent Kits, Diagnostic, Reproducibility of Results, Sensitivity and Specificity, Toxoplasma genetics, DNA, Protozoan isolation & purification, Polymerase Chain Reaction methods, Toxoplasma isolation & purification, Toxoplasmosis parasitology
- Abstract
The aims of the present study were to design an easy and sensitive DNA amplification method for detection of Toxoplasma gondii with low risk of accidental contamination, and to find a rapid method for purification of clinical samples containing potential inhibitors of the amplification reaction. With a pair of primers amplifying a 619-bp fragment of the B1 gene of this parasite it was possible to detect DNA equivalent to 10 parasites. When a third primer was added to the same tube, sensitivity increased to 0.1 parasite. In a comparison of different DNA purification methods, the High Pure PCR Template Preparation Kit (Boehringer Mannheim, Germany) gave the best results. With this purification method and the one-tube hemi-nested PCR, T. gondii DNA was detected in 14 (87.5%) of 16 clinical specimens (amniotic fluid, broncho-alveolar lavage, bone marrow, blood, liver biopsy) in which the parasite was demonstrated by cell culture.
- Published
- 1999
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