Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid.
Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.
Kasper
David C
DC
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna 1090, Austria. david.kasper@meduniwien.ac.at
Sadeghi
Kambis
K
Prusa
Andrea-Romana
AR
Reischer
Georg H
GH
Kratochwill
Klaus
K
Förster-Waldl
Elisabeth
E
Gerstl
Nicole
N
Hayde
Michael
M
Pollak
Arnold
A
Herkner
Kurt R
KR
D000653:Amniotic Fluid / Q000382:microbiology*; D000704:Analysis of Variance; D000818:Animals; D001202:Ascitic Fluid / Q000382:microbiology; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D017125:Genes, Protozoan / Q000235:genetics; D006801:Humans; D051379:Mice; D010533:Peritoneal Lavage / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D011247:Pregnancy; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014125:Toxoplasmosis, Congenital / Q000382:microbiology*
D016054:DNA, Protozoan