title: Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.
abstract: During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors.
authors: Aix-Marseille Université, Faculté de Médecine, UMR MD3, 13284 Marseille, France - APHM, Hôpital de la Timone, Laboratoire de Parasitologie-Mycologie, 13385 Marseille, France.
authors: Fakhri
authors: F
authors: Jeddi
authors: Aix-Marseille Université, Faculté de Médecine, UMR MD3, 13284 Marseille, France - APHM, Hôpital de la Timone, Laboratoire de Parasitologie-Mycologie, 13385 Marseille, France.
authors: Renaud
authors: R
authors: Piarroux
authors: Aix-Marseille Université, Faculté de Médecine, UMR MD3, 13284 Marseille, France - APHM, Hôpital de la Timone, Laboratoire de Parasitologie-Mycologie, 13385 Marseille, France.
authors: Charles
authors: C
authors: Mary
mesh_terms: D001228:Aspergillosis / Q000175:diagnosis; D001232:Aspergillus fumigatus / Q000235:genetics; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002176:Candida albicans / Q000235:genetics; D002177:Candidiasis / Q000175:diagnosis; D002460:Cell Line; D003457:Cryptosporidiosis / Q000175:diagnosis; D003458:Cryptosporidium / Q000235:genetics; D004271:DNA, Fungal / Q000097:blood / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000097:blood / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology; D005872:Giardia / Q000235:genetics; D005873:Giardiasis / Q000175:diagnosis; D006801:Humans; D018314:Leishmania infantum / Q000235:genetics; D007896:Leishmaniasis / Q000175:diagnosis; D009181:Mycoses / Q000175:diagnosis*; D011528:Protozoan Infections / Q000175:diagnosis*
chemical_list: D004271:DNA, Fungal; D016054:DNA, Protozoan