Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation.

Autor: Nourrisson C; Parasitology-Mycology Department, CHU Clermont-Ferrand, 3IHP, 58 Rue Montalembert, 63000, Clermont-Ferrand, France.; Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, M2iSH, UMR INSERM 1071, Clermont Auvergne University, Clermont-Ferrand, France.; National Reference Center (NRC) for Cyptosporidiosis, Microsporidia and Other Digestive Protozoa, Clermont-Ferrand, France., Moniot M; Parasitology-Mycology Department, CHU Clermont-Ferrand, 3IHP, 58 Rue Montalembert, 63000, Clermont-Ferrand, France.; National Reference Center (NRC) for Cyptosporidiosis, Microsporidia and Other Digestive Protozoa, Clermont-Ferrand, France., Tressol M; Parasitology-Mycology Department, CHU Clermont-Ferrand, 3IHP, 58 Rue Montalembert, 63000, Clermont-Ferrand, France., Lambert C; Biostatistics Unit, DRCI, CHU Clermont-Ferrand, Clermont-Ferrand, France., Fréalle E; Laboratory of Parasitology-Mycology, CHU Lille, 59000, Lille, France., Robert-Gangneux F; CHU Rennes, Inserm, EHESP, Irset, UMR_S 1085, Univ Rennes, Rennes, France., Costa D; Laboratory of Parasitology-Mycology, EA7510 ESCAPE, University Hospital of Rouen, Univ Rouen Normandie, Normandy, France.; National Reference Center (NRC) for Cyptosporidiosis, Microsporidia and Other Digestive Protozoa, Rouen, France., Basmaciyan L; Parasitology-Mycology Department, CHU Dijon, Dijon, France.; National Reference Center (NRC) for Cyptosporidiosis, Microsporidia and Other Digestive Protozoa, Dijon, France., Poirier P; Parasitology-Mycology Department, CHU Clermont-Ferrand, 3IHP, 58 Rue Montalembert, 63000, Clermont-Ferrand, France. ppoirier@chu-clermontferrand.fr.; Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, M2iSH, UMR INSERM 1071, Clermont Auvergne University, Clermont-Ferrand, France. ppoirier@chu-clermontferrand.fr.; National Reference Center (NRC) for Cyptosporidiosis, Microsporidia and Other Digestive Protozoa, Clermont-Ferrand, France. ppoirier@chu-clermontferrand.fr.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2024 Jul 04; Vol. 14 (1), pp. 15404. Date of Electronic Publication: 2024 Jul 04.
DOI: 10.1038/s41598-024-66154-2
Abstrakt: Nowadays, the use of qPCR for the diagnosis of intestinal microsporidiosis is increasing. There are several studies on the evaluation of qPCR performance but very few focus on the stool pretreatment step before DNA extraction, which is nevertheless a crucial step. This study focuses on the mechanical pretreatment of stools for Enterocytozoon bieneusi spores DNA extraction. Firstly, a multicenter comparative study was conducted evaluating seven extraction methods (manual or automated) including various mechanical pretreatment. Secondly, several durations and grinding speeds and types of beads were tested in order to optimize mechanical pretreatment. Extraction methods of the various centers had widely-varying performances especially for samples with low microsporidia loads. Nuclisens® easyMAG (BioMérieux) and Quick DNA Fecal/Soil Microbe Microprep kit (ZymoResearch) presented the best performances (highest frequencies of detection of low spore concentrations and lowest Ct values). Optimal performances of mechanical pretreatment were obtained by applying a speed of 30 Hz during 60 s with the TissueLyser II (Qiagen) using commercial beads of various materials and sizes (from ZymoResearch or MP Biomedicals). Overall, the optimal DNA extraction method for E. bieneusi spores contained in stool samples was obtained with a strong but short bead beating using small-sized beads from various materials.
(© 2024. The Author(s).)
Databáze: MEDLINE
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