title: Optimization of Toxoplasma gondii DNA extraction from amniotic fluid using NucliSENS easyMAG and comparison with QIAamp DNA minikit.
abstract: Antenatal diagnosis of congenital toxoplasmosis relies on PCR in amniotic fluid. Because parasitic load is often low, DNA extraction must be optimized. Manual methods remain widespread although automated methods appear more effective. This study aimed at optimizing an automated method and at comparing it with a widespread manual method: QIAamp DNA minikit. To optimize NucliSens easyMAG, we evaluated the addition of proteinase K pre-treatment and the increase of the amount of silica particles used for the extraction. The optimized method was then compared to QIAamp DNA minikit on samples containing less than 25 tachyzoites/ml. NucliSens easyMAG DNA yield was improved after proteinase K pre-treatment (p < 0.01), but not with a higher silica particle input. The optimized method yielded more positive PCRs than the manual method, especially for samples containing 5 tachyzoites/ml or less (71% vs 26%, p < 10(-4)). The DNA amount in samples found positive by PCR was higher after optimized automated extraction than after manual extraction (p < 10(-4)). Proteinase K pre-treatment should be added to extract DNA from amniotic fluid using NucliSens easyMAG. Using this optimized automated method rather than manual methods would improve the sensitivity of Toxoplasma PCR and simplify the daily workflow.
authors: Laboratoire de Parasitologie-Mycologie, Centre Hospitalo-Universitaire de la Timone, 264 rue St-Pierre, 13385, Marseille CEDEX 05, France. benoit.faucher@ap-hm.fr
authors: B
authors: B
authors: Faucher
authors: F
authors: F
authors: Miermont
authors: S
authors: S
authors: Ranque
authors: J
authors: J
authors: Franck
authors: R
authors: R
authors: Piarroux
mesh_terms: D000653:Amniotic Fluid / Q000469:parasitology*; D001331:Automation / Q000379:methods; D016054:DNA, Protozoan / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D014122:Toxoplasma / Q000235:genetics*; D014125:Toxoplasmosis, Congenital / Q000175:diagnosis*
chemical_list: D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic