Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.
OBJECTIVE
In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis.


METHODS
We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection.


RESULTS
The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs.


CONCLUSIONS
Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.
Calderaro
Adriana
A
Faculty of Medicine and Surgery, Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma, Parma, Italy. adriana.calderaro@unipr.it
Piccolo
Giovanna
G
Gorrini
Chiara
C
Peruzzi
Simona
S
Zerbini
Laura
L
Bommezzadri
Simona
S
Dettori
Giuseppe
G
Chezzi
Carla
C
D000328:Adult; D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D017125:Genes, Protozoan; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics* / Q000302:isolation & purification; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000469:parasitology
D016054:DNA, Protozoan