Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid.
Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.
Yera
H
H
Hôpital Cochin AP-HP, Université Paris Descartes, Parasitology-Mycology Laboratory, Paris, France.
Filisetti
D
D
Bastien
P
P
Ancelle
T
T
Thulliez
P
P
Delhaes
L
L
D000653:Amniotic Fluid / Q000469:parasitology*; D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014123:Toxoplasmosis / Q000175:diagnosis* / Q000469:parasitology
D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase