An alternative DNA extraction method for detection of Blastocystis spp. in human fecal samples

Karasartova, Djursun; Güreser, Ayşe Semra; Ruh, Emrah; Turegun-Atasoy, Buse; Calgin, Mustafa Kerem; Taşçi, Leyla; Taylan-Özkan, Ayşegül

doi:10.1016/j.exppara.2018.01.019

Cited by 3 publications

(4 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, both agarose-gel electrophoresis image (Figure 2) and OD 260 measurement indicated that BB greatly enhanced (P = 0.001; Table 1) DNA yield. Previous studies also indicate that BB effectively increases DNA yield in fecal samples (de Boer et al, 2010;Karasartova et al, 2018). Furthermore, the lack of interaction between CL and BB was another important finding of this study (P ≥ 0.48, Table 1), which indicates that the improved DNA yield caused by BB is independently on CL.…”

Section: Dna Yieldsupporting

confidence: 74%

“…Furthermore, sand is cheaper and more available than bead; replacing bead with sand may decrease the costs for DNA extraction. Including sand sufficiently extracts DNA from human feces for detecting pathogens (Karasartova et al, 2018). However, no study has been performed to evaluate the effect of sand beater on the efficiency of DNA extraction from rumen microorganisms.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa

Zhang

Wang

et al. 2020

Front. Microbiol.

View full text Add to dashboard Cite

QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, United States) and RBB + C ( Yu and Morrison, 2004 ) methodologies are widely employed to extract microbial DNA from rumen samples and can exhibit different efficiencies of obtaining DNA yield, quality, and downstream amplicon sequence analysis. No study has conducted to investigate the contributions of chemical and mechanical lysis on DNA extraction, which included chemical lysis from QIAamp Fast DNA Stool Mini Kit (QIA) and RBB + C (YM), bead (BB), and sand beating (SB). Effects of chemical lysis and bead beating (BB) were investigated by conducting a 2 × 2 factorial-designed experiment with four methodologies, including QIA without (QIA−) and with BB (QIA + BB), and YM without (YM−) and with BB (YM + BB). Comparisons between bead and sand were conducted by comparing methodologies of YM + BB and YM + SB. Comparing with QIA, YM had lower ( P ≤ 0.10) OD 260 / 280 and diversity of ZOTUs and length polymorphism of protozoal amplicons but harvested greater ( P ≤ 0.086) DNA from fibrolytic bacteria such as Ruminococcaceae lineages. Including BB increased ( P = 0.001) total DNA yield without affecting ( P ≥ 0.55) OD 260 / 280 and richness of bacterial ZOTUs but decreased ( P ≤ 0.08) richness of both ZOTUs and length polymorphism of protozoal amplicon. Bead beating and SB showed no difference ( P ≥ 0.19) in DNA yield and quality and bacterial and protozoal community. In summary, chemical lysis provided by RBB + C and QIAamp Fast DNA Stool Mini Kit should be better to extract DNA for analyzing bacterial and protozoal community, respectively. Sand can be an alternative beater for DNA extraction, and mechanical lysis is not recommended for protozoal community analysis.

show abstract

Section: Dna Yieldsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa

Zhang

Wang

et al. 2020

Front. Microbiol.

View full text Add to dashboard Cite

show abstract

“…Whilst emulsion PCR may help to prevent chimera formation, this was not used in our study and future work using this technique should be considered. Although recent studies have shown that chemical lysis is not inferior to mechanical lysis in rumen samples and from human saliva [ 16 , 22 ], human gut samples have consistently shown that mechanical lysis or a combination of enzymatic and mechanical disruption results in higher degrees of microbial diversity and have the greatest effect on gut microbiome composition [ 17 , 23 , 24 , 25 ]. Furthermore, our results demonstrated that between the two methods, there was considerable variation in relative abundances within Gram-positive bacteria but similar abundance levels in the Gram-negative Bacteroidota phylum.…”

Section: Discussionmentioning

confidence: 99%

Variability in the Pre-Analytical Stages Influences Microbiome Laboratory Analyses

Kumar

Gravdal

Segal

et al. 2022

Genes

View full text Add to dashboard Cite

Introduction: There are numerous confounding variables in the pre-analytical steps in the analysis of gut microbial composition that affect data consistency and reproducibility. This study compared two DNA extraction methods from the same faecal samples to analyse differences in microbial composition. Methods: DNA was extracted from 20 faecal samples using either (A) chemical/enzymatic heat lysis (lysis buffer, proteinase K, 95 °C + 70 °C) or (B) mechanical and chemical/enzymatic heat lysis (bead-beating, lysis buffer, proteinase K, 65 °C). Gut microbiota was mapped through the 16S rRNA gene (V3-V9) using a set of pre-selected DNA probes targeting >300 bacteria on different taxonomic levels. Apart from the pre-analytical DNA extraction technique, all other parameters including microbial analysis remained the same. Bacterial abundance and deviations in the microbiome were compared between the two methods. Results: Significant variation in bacterial abundance was seen between the different DNA extraction techniques, with a higher yield of species noted in the combined mechanical and heat lysis technique (B). The five predominant bacteria seen in both (A) and (B) were Bacteroidota spp. and Prevotella spp. (p = NS), followed by Bacillota (p = 0.005), Lachhnospiraceae (p = 0.0001), Veillonella spp. (p < 0.0001) and Clostridioides (p < 0.0001). Conclusion: As microbial testing becomes more easily and commercially accessible, a unified international consensus for optimal sampling and DNA isolation procedures must be implemented for robustness and reproducibility of the results.

show abstract

“…DNA purification from stool was carried out according to the protocols [55]. In brief, the stools from the piglets were collected in storage tubes and then stored in liquid nitrogen until DNA extraction.…”

Section: Methodsmentioning

confidence: 99%

Effect of Baicalin-Aluminum Complexes on Fecal Microbiome in Piglets

Zhuang

Li-wei

et al. 2019

IJMS

View full text Add to dashboard Cite

The gut microbiome has important effects on gastrointestinal diseases. Diarrhea attenuation functions of baicalin (BA) is not clear. Baicalin–aluminum complexes (BBA) were synthesized from BA, but the BBA’s efficacy on the diarrhea of piglets and the gut microbiomes have not been explored and the mechanism remains unclear. This study has explored whether BBA could modulate the composition of the gut microbiomes of piglets during diarrhea. The results showed that the diarrhea rate reduced significantly after treatment with BBA. BBA altered the overall structure of the gut microbiomes. In addition, the Gene Ontology (GO) enrichment analysis indicated that the functional differentially expressed genes, which were involved in the top 30 GO enrichments, were associated with hydrogenase (acceptor) activity, nicotinamide-nucleotide adenylyltransferase activity, and isocitrate lyase activity, belong to the molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that flagellar assembly, bacterial chemotaxis, lipopolysaccharide biosynthesis, ATP-binding cassette transporters (ABC) transporters, biosynthesis of amino acids, and phosphotransferase system (PTS) were the most enriched during BBA treatment process. Taken together, our results first demonstrated that BBA treatment could modulate the gut microbiomes composition of piglets with diarrhea, which may provide new potential insights on the mechanisms of gut microbiomes associated underlying the antimicrobial efficacy of BBA.

show abstract

An alternative DNA extraction method for detection of Blastocystis spp. in human fecal samples

Cited by 3 publications

References 46 publications

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa

Effects of Chemical and Mechanical Lysis on Microbial DNA Yield, Integrity, and Downstream Amplicon Sequencing of Rumen Bacteria and Protozoa

Variability in the Pre-Analytical Stages Influences Microbiome Laboratory Analyses

Effect of Baicalin-Aluminum Complexes on Fecal Microbiome in Piglets

Contact Info

Product

Resources

About