title: Design of a one-tube hemi-nested PCR for detection of Toxoplasma gondii and comparison of three DNA purification methods.
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.
authors.affiliation: Servei de Microbiologia.
authors.forename: Marc
authors.initials: M
authors.lastname: Pujol-Riqué
authors.affiliation: Laboratoire de Parasitologie-Mycologie, Hospital Saint-Louis, Paris, France.
authors.forename: Francis
authors.initials: F
authors.lastname: Derouin
authors.affiliation: Servei de Microbiologia.
authors.forename: Alberto
authors.initials: A
authors.lastname: Garcia-Quintanilla
authors.affiliation: Servei de Microbiologia.
authors.forename: M E
authors.initials: ME
authors.lastname: Valls
authors.affiliation: Servei de Malalties Infeccioses, IDIBAPS, Hospital Clinic Universitari de Barcelona. Facultat de Medicina, Universitat de Barcelona, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Miró
authors.affiliation: Servei de Microbiologia.
authors.forename: M T
authors.initials: MT
authors.lastname: Jiménez De Anta
mesh_terms: D000818:Animals; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification*; D005784:Gene Amplification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000469:parasitology*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic