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

The aims of the present study were to design an easy and sensitive DNA ampl ification method for detection of Toxoplasma gondii with low risk of accide ntal contamination, and to find a rapid method for purification of clinical samples containing potential inhibitors of the amplification reaction, Wit h a pair of primers amplifying a 619-bp fragment of the B1 gene of this par asite it was possible to detect DNA equivalent to 10 parasites. When a thir d 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 T emplate Preparation Kit (Boehringer Mannheim, Germany) gave the best result s, With this purification method and the one-tube hemi-nested PCR, T. gondi i DNA was detected in 14 (87.5%) of 16 clinical specimens (amniotic fluid, broncho-alveolar lavage, bone marrow, blood, liver biopsy) in which the par asite was demonstrated by cell culture.