JCM Accepts, published online ahead of print on 21 October 2009

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J. Clin. Microbiol. doi:10.1128/JCM.01164-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Multicentre comparative evaluation of five commercial methods for Toxoplasma DNA extraction from amniotic fluid

H Yera, D Filisetti, P Bastien, T Ancelle, P Thulliez, and L Delhaes^*

Hôpital Cochin AP-HP, Université Paris Descartes, Parasitology-Mycology laboratory, Paris, France; Institut de Puériculture et de Périnatalogie, Laboratoire de la Toxoplasmose, Paris, France; Hôpitaux Universitaires de Strasbourg/Université de Strasbourg, Laboratoire de Parasitologie et Mycologie Médicale, Strasbourg, France; Centre Hospitalier Universitaire de Montpellier/Université Montpellier 1, Laboratoire de Parasitologie-Mycologie, Montpellier, France; Parasitology-Mycology Service (EA3609) Faculty of Medicine, Univ Nord de France (UDSL), University Hospital Centre & IFR-142, Institut Pasteur de Lille, France

^* To whom correspondence should be addressed. Email: l-delhaes{at}chru-lille.fr.

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 commercialised manual extraction methods.

The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen) and EasyMag (BioMérieux) automated procedures were compared to the two manual DNA extraction kits, the QIAamp DNA Mini Kit (Qiagen) and the High Pure PCR Template Preparation Kit (Roche). Evaluation was carried out two specific Toxoplasma-PCRs (targeting the 529-bp repeat element), inhibitor search-PCRs and human beta-globin-PCRs. The samples consisted in 4 ml of amniotic fluid with or without a calibrated Toxoplasma 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 FRET probes. A total of 1178 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/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF (< 5 Toxoplasma/ml), a difference that might have repercussions since low parasite concentrations in amniotic fluid and can lead to congenital toxoplasmosis.