ArticleBMC research notes2013
A semi-automated protocol for Archaea DNA extraction from stools.
Article in BMC research notes, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
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- Interpreting tree ensemble machine learning models with endoR.PLoS computational biology · 2022Article
- New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections-A Narrative Mini-Review on Challenges in the Tropics.Tropical medicine and infectious disease · 2021Review
- Detection of Methanobrevibacter smithii in vaginal samples collected from women diagnosed with bacterial vaginosis.European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2019Article
- Co-culture of Methanobrevibacter smithii with enterobacteria during urinary infection.EBioMedicine · 2019Article
- Methanogens as emerging pathogens in anaerobic abscesses.European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2019Review
- Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.BMC research notes · 2018Article
- Comparison of Infant Gut and Skin Microbiota, Resistome and Virulome Between Neonatal Intensive Care Unit (NICU) Environments.Frontiers in microbiology · 2018Article
- Collection of non-meconium stool on fecal occult blood cards is an effective method for fecal microbiota studies in infants.Microbiome · 2017Article
- Comparison of an automated nucleic acid extraction system with the column-based procedure.European journal of microbiology & immunology · 2015Article
- Current and past strategies for bacterial culture in clinical microbiology.Clinical microbiology reviews · 2015Review
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Authors and funding
4 authors.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundThe PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens.
findingsWe tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P = 0.001) with an average Ct value of 27.4 (P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens (P = 0.001) with an average Ct value of 30.33 (P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semi-automated protocol (P > 0.1 and P > 0.1).
conclusionProteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification.
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