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 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.
Pujol-Riqué
Marc
M
Servei de Microbiologia.
Derouin
Francis
F
Laboratoire de Parasitologie-Mycologie, Hospital Saint-Louis, Paris, France.
Garcia-Quintanilla
Alberto
A
Servei de Microbiologia.
Valls
M E
ME
Servei de Microbiologia.
Miró
J M
JM
Servei de Malalties Infeccioses, IDIBAPS, Hospital Clinic Universitari de Barcelona. Facultat de Medicina, Universitat de Barcelona, Spain.
Jiménez De Anta
M T
MT
Servei de Microbiologia.
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*
D017931:DNA Primers; D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic