A semi-automated protocol for Archaea DNA extraction from stools

Khelaifia, Saber; Ramonet, Pierre-Yves; Buffet, Marielle Bedotto; Drancourt, Michel

doi:10.1186/1756-0500-6-186

Cited by 17 publications

(15 citation statements)

References 21 publications

(31 reference statements)

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“…Those approaches comprise virus diagnostics for cytomegalovirus [13,16,20], hepatitis virus B and C [18], human immunodefi ciency virus [12,18], PCRs for bacterial [15] and viral [4,15] respiratory pathogens, stool pathogens [14] or commensalic bacteria from stool [7], and biothreat agents [5], as well as PCR diagnostics for parasitic diseases like toxoplasmosis [17] and for fungal pathogens like Aspergillus fumigatus [3]. EZ1 extraction is affected by various preanalytic factors like sample type and chosen preprocessing protocol In-house according to [29] n.a.…”

Section: Discussionmentioning

confidence: 99%

Comparison of an automated nucleic acid extraction system with the column-based procedure

Frickmann¹,

Hinz²,

Hagen³

2015

European Journal of Microbiology and Immunology

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Here, we assessed the extraction efficiency of a deployable bench-top nucleic acid extractor EZ1 in comparison to the column-based approach with complex sample matrices.A total of 48 EDTA blood samples and 81 stool samples were extracted by EZ1 automated extraction and the column-based QIAamp DNA Mini Kit. Blood sample extractions were assessed by two real-time malaria PCRs, while stool samples were analyzed by six multiplex real-time PCR assays targeting bacterial, viral, and parasitic stool pathogens. Inhibition control PCR testing was performed as well.In total, 147 concordant and 13 discordant pathogen-specific PCR results were obtained. The latter comprised 11 positive results after column-based extraction only and two positive results after EZ1 extraction only. EZ1 extraction showed a higher frequency of inhibition. This phenomenon was, however, inconsistent for the different PCR schemes. In case of concordant PCR results, relevant differences of cycle threshold numbers for the compared extraction schemes were not observed.Switches from well-established column-based extraction to extraction with the automated EZ1 system do not lead to a relevantly reduced yield of target DNA when complex sample matrices are used. If sample inhibition is observed, column-based extraction from another sample aliquot may be considered.

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Section: Discussionmentioning

confidence: 99%

Comparison of an automated nucleic acid extraction system with the column-based procedure

Frickmann¹,

Hinz²,

Hagen³

2015

European Journal of Microbiology and Immunology

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“…Samples were placed on a shaking heat block at 95 °C for 5 min at 2000 rpm, cooled on ice for 2–5 min, and centrifuged at 20,000× g for 2 min. Supernatant was removed, avoiding the pellet, and placed in a new 2-mL ET [ 8 , 10 , 11 ] with one Inhibit X tablet (Qiagen, Valencia, CA). Samples were vortexed until the tablet was dissolved and incubated at room temperature for 3 min [ 11 , 14 ].…”

Section: Methodsmentioning

confidence: 99%

“…Literature suggested that the EZ1 tissue kit in conjunction with the bacteria extraction card produced suitable DNA yields for sequencing [ 10 , 11 ]. Ideally, an automated workflow is preferred for extraction because of the possibility of high sample volumes in future studies.…”

Section: Methodsmentioning

confidence: 99%

Collection of non-meconium stool on fecal occult blood cards is an effective method for fecal microbiota studies in infants

Wong¹,

Clemency²,

Klein³

et al. 2017

Microbiome

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BackgroundEffective methods are needed to collect fecal samples from children for large-scale microbiota studies. Stool collected on fecal occult blood test (FOBT) cards that can be mailed provides an effective solution; however, the quality of sequencing resulting from this method is unknown.The aim of this study is to compare microbiota metrics of 16S ribosomal RNA (rRNA) gene sequencing from stool and meconium collected on FOBT cards with stool collected in an Eppendorf tube (ET) under different conditions.MethodsEight stool samples from children in diapers aged 0 month–2 years and three meconium samples were collected and stored as follows: (1) ≤ 2 days at room temperature (RT) in an ET, (2) 7 days at − 80 °C in an ET, (3) 3–5 days at RT on a FOBT card, (4) 7 days at RT on a FOBT card, and (5) 7 days at − 80 °C on a FOBT card. Samples stored at − 80 °C were frozen immediately. Each specimen/condition underwent 16S rRNA gene sequencing with replicates on the Illumina MiSeq. Alpha and beta diversity measures and relative abundance of major phyla were compared between storage conditions and container (ET vs. FOBT card), with pairwise comparison between different storage conditions and the “standard” of 7 days at − 80 °C in an ET and fresh stool in an ET.ResultsStool samples clustered mainly by individual diaper (P < 10−5, Adonis), rather than by storage condition (P = 0.42) or container (P = 0.16). However, meconium samples clustered more by container (P = 0.002) than by individual diaper (P = 0.009) and storage condition (P = 0.02).Additionally, there were no differences in alpha diversity measures and relative abundance of major phyla after Bonferroni correction between stool stored on a FOBT card at RT for 7 days with stool stored in an ET tube at − 80 °C; differences in alpha diversity were seen however when compared to fresh stool in an ET. Overall, based on the intraclass correlation coefficient (ICC), the different storage containers/conditions are reliable in preserving the microbial memberships and slightly less reliable in preserving the alpha diversity and relative microbial composition of infant stool.ConclusionsAcknowledging certain limitations, FOBT cards may be a useful tool in large-scale stool microbiota studies in children requiring outpatient follow-up where only small amounts of stool can be obtained, but should not be used when studying meconium.Electronic supplementary materialThe online version of this article (10.1186/s40168-017-0333-z) contains supplementary material, which is available to authorized users.

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“…A universal, reproducible, simple, efficient and robust extraction method is particularly valuable for PCR-based diagnosis which is increasingly used in both clinical laboratories and epidemiological studies [ 9 , 12 , 13 ]. To date, the EZ1 ® procedure has been validated for the DNA extraction of viruses and bacteria [ 14 ] but also for fastidious microorganisms such as archaea [ 15 ]. Therefore, and in line with our findings, we recommend using the EZ1 ® kit-based procedure, as described herein, for the PCR-based detection of eukaryotic intestinal pathogens in stool samples.…”

Section: Main Textmentioning

confidence: 99%

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

Mary

Toga

et al. 2018

BMC Res Notes

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ObjectiveEfficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi.ResultsWhereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

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A semi-automated protocol for Archaea DNA extraction from stools

Cited by 17 publications

References 21 publications

Comparison of an automated nucleic acid extraction system with the column-based procedure

Comparison of an automated nucleic acid extraction system with the column-based procedure

Collection of non-meconium stool on fecal occult blood cards is an effective method for fecal microbiota studies in infants

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples

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