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The Journal of Medical Microbiology, Vol 48, Issue 9 857-862, Copyright © 1999 by Society for General Microbiology

JOURNAL ARTICLE

Design of a one-tube hemi-nested PCR for detection of Toxoplasma gondii and comparison of three DNA purification methods

M. Pujol-Rique, F. Derouin, A. Garcia-Quintanilla, M. E. Valls, J. M. Miro and M. T. Jimenez de Anta
IDIBAPS, Hospital Clinic Universitari de Barcelona. Facultat de Medicina, Universitat de Barcelona, Spain. pujol@medicina.ub.es

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.


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