Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
04 Jul 2024
04 Jul 2024
Historique:
received:
12
07
2023
accepted:
27
06
2024
medline:
5
7
2024
pubmed:
5
7
2024
entrez:
4
7
2024
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 38965323
doi: 10.1038/s41598-024-66154-2
pii: 10.1038/s41598-024-66154-2
doi:
Substances chimiques
DNA, Fungal
0
Types de publication
Journal Article
Multicenter Study
Comparative Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
15404Informations de copyright
© 2024. The Author(s).
Références
Autier, B., Gangneux, J.-P. & Robert-Gangneux, F. Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis. Parasite Paris Fr. 28, 33 (2021).
doi: 10.1051/parasite/2021034
Morio, F. et al. Assessment of the first commercial multiplex PCR kit (ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples. Diagn. Microbiol. Infect. Dis. 95, 34–37 (2019).
doi: 10.1016/j.diagmicrobio.2019.04.004
pubmed: 31079868
Argy, N. et al. Selecting a multiplex PCR panel for accurate molecular diagnosis of intestinal protists: a comparative study of Allplex® (Seegene®), G-DiaParaTrio (Diagenode®), and RIDA®GENE (R-Biopharm®) assays and microscopic examination. Parasite Paris Fr. 29, 5 (2022).
doi: 10.1051/parasite/2022003
Hartuis, S. et al. The Novodiag® Stool parasites assay, an innovative high-plex technique for fast detection of protozoa, helminths and microsporidia in stool samples: a retrospective and prospective study. Parasite Paris Fr. 29, 27 (2022).
doi: 10.1051/parasite/2022026
Claudel, L. et al. Comparative study of eleven mechanical pretreatment protocols for cryptosporidium parvum DNA extraction from stool samples. Microorganisms 9, 297 (2021).
doi: 10.3390/microorganisms9020297
pubmed: 33540520
pmcid: 7912823
Valeix, N. et al. Multicenter comparative study of six cryptosporidium parvum DNA extraction protocols including mechanical pretreatment from stool samples. Microorganisms 8, E1450 (2020).
doi: 10.3390/microorganisms8091450
Han, B., Pan, G. & Weiss, L. M. Microsporidiosis in humans. Clin. Microbiol. Rev. 34, e0001020. https://doi.org/10.1128/CMR.00010-20 (2021).
doi: 10.1128/CMR.00010-20
pubmed: 34190570
Polley, S. D., Boadi, S., Watson, J., Curry, A. & Chiodini, P. L. Y. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J. Med. Microbiol. 60, 459–466 (2011).
doi: 10.1099/jmm.0.026781-0
pubmed: 21183599
Wolk, D. M., Schneider, S. K., Wengenack, N. L., Sloan, L. M. & Rosenblatt, J. E. Real-time PCR method for detection of encephalitozoonintestinalis from Stool specimens. J. Clin. Microbiol. 40, 3922–3928 (2002).
doi: 10.1128/JCM.40.11.3922-3928.2002
pubmed: 12409353
pmcid: 139654
Halstead, F. D. et al. Universal extraction method for gastrointestinal pathogens. J. Med. Microbiol. 62, 1535–1539 (2013).
doi: 10.1099/jmm.0.058743-0
pubmed: 23831766
Ariefdjohan, M. W., Savaiano, D. A. & Nakatsu, C. H. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutr. J. 9, 23 (2010).
doi: 10.1186/1475-2891-9-23
pubmed: 20492702
pmcid: 2901363
Babaei, Z., Oormazdi, H., Rezaie, S., Rezaeian, M. & Razmjou, E. Giardia intestinalis: DNA extraction approaches to improve PCR results. Exp. Parasitol. 128, 159–162 (2011).
doi: 10.1016/j.exppara.2011.02.001
pubmed: 21315715
Paulos, S. et al. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp, Giardia duodenalis, and Entamoeba spp.. J. Microbiol. Methods 127, 68–73 (2016).
doi: 10.1016/j.mimet.2016.05.020
pubmed: 27241828
Hawash, Y. DNA extraction from protozoan oocysts/cysts in feces for diagnostic PCR. Korean J. Parasitol. 52, 263–271 (2014).
doi: 10.3347/kjp.2014.52.3.263
pubmed: 25031466
pmcid: 4096637
Çetinkaya, Ü., Charyyeva, A., Sivcan, E. & Gürbüz, E. Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation. Acta Parasitol. 63, 386–392 (2018).
doi: 10.1515/ap-2018-0044
pubmed: 29654668
Menu, E. et al. Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples. BMC Res. Notes 11, 206 (2018).
doi: 10.1186/s13104-018-3300-2
pubmed: 29587846
pmcid: 5869780
Yera, H. et al. Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 24(1100), e7-1100.e11 (2018).
Jeddi, F., Piarroux, R. & Mary, C. Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases. Parasite Paris Fr. 20, 52 (2013).
doi: 10.1051/parasite/2013051
Le Govic, Y. et al. Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk. Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol. 35, 137–148 (2016).
doi: 10.1007/s10096-015-2519-2
You, M.-J. Effects of different sizes of glass beads on the release of sporocysts from Eimeria tenella oocysts. Korean J. Parasitol. 52, 317–319 (2014).
doi: 10.3347/kjp.2014.52.3.317
pubmed: 25031475
pmcid: 4096646
Cha, J.-O., Talha, A. F. S. M., Lim, C. W. & Kim, B. Effects of glass bead size, vortexing speed and duration on Eimeria acervulina oocyst excystation. Exp. Parasitol. 138, 18–24 (2014).
doi: 10.1016/j.exppara.2014.01.001
pubmed: 24457170
Moniot, M. et al. Assessment of a multiplex PCR for the simultaneous diagnosis of intestinal cryptosporidiosis and microsporidiosis: epidemiologic report from a French prospective study. J. Mol. Diagn. JMD 23, 417–423 (2021).
doi: 10.1016/j.jmoldx.2020.12.005
pubmed: 33387699
Cohen, J. Statistical power analysis for the behavioral sciences. (1988).