Comparison of the utility of five commercial kits for extraction of DNA from Aspergillus fumigatus spores.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Urszula Nawrot ◽  
Katarzyna Wlodarczyk ◽  
Magdalena Wrobel ◽  
Anita Wasik ◽  
Tadeusz Dobosz
Keyword(s):  
Dna Extraction ◽  
Enzymatic Digestion ◽  
Cell Disruption ◽  
Clinical Samples ◽  
Rrna Gene ◽  
28S Rrna ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Dna Yield ◽  
Commercial Kits

The aim of this study was to compare the efficiency of DNA extraction from water as well as from blood samples spiked with A. fumigatus spores, using selected commercial kits. Extraction of DNA according to manufacturer's protocols was preceded by blood cells lysis and disruption of fungal cells by enzymatic digestion or bead beating. The efficiency of DNA extraction was measured by PCR using Aspergillus-specific primers and SYBR Green I dye or TaqMan probes targeting 28S rRNA gene. All methods allowed the detection of Aspergillus at the lowest tested density of water suspensions of spores (10¹ cells/ml). The highest DNA yield was obtained using the ZR Fungal/Bacterial DNA kit, YeastStar Genomic DNA kit, and QIAamp DNA Mini kit with mechanical cell disruption. The ZR Fungal/Bacterial DNA and YeastStar kits showed the highest sensitivity in examination of blood samples spiked with Aspergillus (100 % for the detection of 10² spores and 75 % for 10¹ spores). Recently, the enzymatic method ceased to be recommended for examination of blood samples for Aspergillus, thus ZR Fungal/Bacterial DNA kit and QIAamp DNA Mini kit with mechanical cell disruption could be used for extraction of Aspergillus DNA from clinical samples.

scholarly journals Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR

2021 ◽  
Author(s):  
Annemarie Siebert ◽  
Katharina Hofmann ◽  
Lena Staib ◽  
Etienne V. Doll ◽  
Siegfried Scherer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Extraction ◽  
Raw Milk ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Analysis ◽  
Eukaryotic Dna ◽  
Selective Lysis ◽  
The Impact

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

scholarly journals Circulating bacterial signature is linked to metabolic disease and shifts with metabolic alleviation after bariatric surgery

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Rima M. Chakaroun ◽  
Lucas Massier ◽  
Anna Heintz-Buschart ◽  
Nedal Said ◽  
Joerg Fallmann ◽  
...  
Keyword(s):  
Metabolic Disease ◽  
Bariatric Surgery ◽  
Weight Loss ◽  
Metabolic Health ◽  
Rrna Gene ◽  
Clinical Group ◽  
Analytical Control ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Living Bacteria

Abstract Background The microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery. Methods Subjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH). Results The contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiometabolic risk parameters. CARD-FISH allowed the detection of living bacteria in blood samples in obesity. Conclusions We show that the circulating bacterial signature reflects metabolic disease and its improvement after bariatric surgery. Our work provides contaminant-aware evidence for the presence of living bacteria in the blood and suggests a putative crosstalk between components of the blood and metabolism in metabolic health regulation.

scholarly journals The Influence of DNA Extraction and Lipid Removal on Human Milk Bacterial Profiles

2020 ◽  
Vol 3 (2) ◽  
pp. 39 ◽  
Author(s):  
Anna Ojo-Okunola ◽  
Shantelle Claassen-Weitz ◽  
Kilaza S. Mwaikono ◽  
Sugnet Gardner-Lubbe ◽  
Heather J. Zar ◽  
...  
Keyword(s):  
Human Milk ◽  
Dna Extraction ◽  
Bacterial Population ◽  
Illumina Miseq ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Dna Recovery ◽  
Culture Independent ◽  
Zymo Research

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

scholarly journals Bacterial DNAemia is associated with serum zonulin levels in older subjects

2020 ◽  
Author(s):  
Giorgio Gargari ◽  
Valentina Taverniti ◽  
Cristian Del Bo’ ◽  
Stefano Bernardi ◽  
Cristina Andres-Lacueva ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Serum Levels ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Gene Copies ◽  
Older Subjects ◽  
Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

scholarly journals Impact of enzymatic digestion on bacterial community composition in CF airway samples

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3362 ◽  
Author(s):  
Kayla M. Williamson ◽  
Brandie D. Wagner ◽  
Charles E. Robertson ◽  
Emily J. Johnson ◽  
Edith T. Zemanick ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Dna Extraction ◽  
Community Composition ◽  
Bacterial Communities ◽  
Beta Diversity ◽  
Diversity Index ◽  
Bacterial Community Composition ◽  
Microbial Community Composition ◽  
Enzymatic Digestion ◽  
Rrna Gene

BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection ofStaphylococcus,an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the generaGemella(p < 0.01),Streptococcus(p < 0.01), andRothia(p < 0.01).Staphylococcus(p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD:Veillonella(p < 0.01),Granulicatella(p < 0.01),Prevotella(p < 0.01), andGemella(p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection ofStaphylococcusin CF OP samples. The enhanced identification ofStaphylococcus aureusis a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities.

scholarly journals Molecular Diagnosis of Invasive Aspergillosis and Detection of Azole Resistance by a Newly Commercialized PCR Kit

2017 ◽  
Vol 55 (11) ◽  
pp. 3210-3218 ◽  
Author(s):  
Eric Dannaoui ◽  
Frédéric Gabriel ◽  
Manuel Gaboyard ◽  
Gaëlle Lagardere ◽  
Lucile Audebert ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Rrna Gene ◽  
28S Rrna ◽  
Serum Samples ◽  
Content Type ◽  
28S Rrna Gene

ABSTRACTAspergillus fumigatusis the main species responsible for aspergillosis in humans. The diagnosis of aspergillosis remains difficult, and the rapid emergence of azole resistance inA. fumigatusis worrisome. The aim of this study was to validate the new MycoGENIEA. fumigatusreal-time PCR kit and to evaluate its performance on clinical samples for the detection ofA. fumigatusand its azole resistance. This multiplex assay detects DNA from theA. fumigatusspecies complex by targeting the multicopy 28S rRNA gene and specific TR34and L98H mutations in the single-copy-numbercyp51Agene ofA. fumigatus. The specificity ofcyp51Amutation detection was assessed by testing DNA samples from 25 wild-type or mutated clinicalA. fumigatusisolates. Clinical validation was performed on 88 respiratory samples obtained from 62 patients and on 69 serum samples obtained from 16 patients with proven or probable aspergillosis and 13 patients without aspergillosis. The limit of detection was <1 copy for theAspergillus28S rRNA gene and 6 copies for thecyp51Agene harboring the TR34and L98H alterations. No cross-reactivity was detected with various fungi and bacteria. All isolates harboring the TR34and L98H mutations were accurately detected by quantitative PCR (qPCR) analysis. With respiratory samples, qPCR results showed a sensitivity and specificity of 92.9% and 90.1%, respectively, while with serum samples, the sensitivity and specificity were 100% and 84.6%, respectively. Our study demonstrated that this new real-time PCR kit enables sensitive and rapid detection ofA. fumigatusDNA and azole resistance due to TR34and L98H mutations in clinical samples.

scholarly journals DNA extraction approaches substantially influence the assessment of the human breast milk microbiome

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chloe A. Douglas ◽  
Kerry L. Ivey ◽  
Lito E. Papanicolas ◽  
Karen P. Best ◽  
Beverly S. Muhlhausler ◽  
...  
Keyword(s):  
Breast Milk ◽  
Dna Extraction ◽  
Infant Development ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Human Breast Milk ◽  
Human Breast ◽  
Dna Yield ◽  
Commensal Microbiota ◽  
Milk Samples

AbstractIn addition to providing nutritional and bioactive factors necessary for infant development, human breast milk contains bacteria that contribute to the establishment of commensal microbiota in the infant. However, the composition of this bacterial community differs considerably between studies. We hypothesised that bacterial DNA extraction methodology from breast milk samples are a substantial contributor to these inter-study differences. We tested this hypothesis by applying five widely employed methodologies to a mock breast milk sample and four individual human breast milk samples. Significant differences in DNA yield and purity were observed between methods (P < 0.05). Microbiota composition, assessed by 16S rRNA gene amplicon sequencing, also differed significantly with extraction methodology (P < 0.05), including in the contribution of contaminant signal. Concerningly, many of the bacterial taxa identified here as contaminants have been reported as components of the breast milk microbiome in other studies. These findings highlight the importance of using stringent, well-validated, DNA extraction methodologies for analysis of the breast milk microbiome, and exercising caution interpreting microbiota data from low-biomass contexts.

scholarly journals Bacterial DNAemia is associated with serum zonulin levels in older subjects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giorgio Gargari ◽  
Giacomo Mantegazza ◽  
Valentina Taverniti ◽  
Cristian Del Bo’ ◽  
Stefano Bernardi ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Serum Levels ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Taxonomic Profiling ◽  
Older Subjects ◽  
Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all samples contained detectable amounts of bacterial DNA with a concentration that varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly associated with the serum levels of zonulin, a marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected from the same subjects. 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of the genus Pseudomonas. Several control samples were also analyzed to assess the influence of contaminant bacterial DNA potentially originating from reagents and materials. The data reported here suggest that para-cellular permeability of epithelial (and, potentially, endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

Microbial Associations of Four Species of Algal Symbiont-bearing Foraminifers from the Florida Reef Tract, Usa

2019 ◽  
Vol 49 (2) ◽  
pp. 178-190
Author(s):  
Makenna M. Martin ◽  
Christina A. Kellogg ◽  
Pamela Hallock
Keyword(s):  
Dna Extraction ◽  
Variable Region ◽  
Florida Keys ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Microbiome Composition ◽  
Algal Symbiont ◽  
Reef Tract ◽  
Bacterial Dna Extraction

Abstract While microbiome research is a rapidly expanding field of study, relatively little is known of the microbiomes associated with Foraminifera. This preliminary study investigated microbes associated with four species of Foraminifera, representing two taxonomic orders, which host three kinds of algal endosymbionts. A major objective was to explore potential influences on the microbiome composition, including phylogenetic relatedness among the host species, similarities in algal symbionts hosted, and environmental conditions from which the specimens were collected. Samples examined from two locations along the middle Florida Keys reef tract included 45 foraminiferal specimens and four environmental samples. Bacterial DNA extraction from individual specimens was followed by amplification and amplicon sequencing of the V4 variable region of the 16S rRNA gene; results were obtained from 21 specimens. The Order Miliolida, Family Soritidae, was represented by 5–8 specimens of each of three species: Archaias angulatus and Cyclorbiculina compressa, which both host chlorophyte symbionts, and Sorites orbiculus, which hosts dinoflagellate symbionts. Three Ar. angulatus specimens from which the microbiome was successfully sequenced shared 177 OTUs. Six C. compressa specimens successfully sequenced shared 58 OTUs, of which 31 were also shared by the three specimens of Ar. angulatus. Four successfully sequenced S. orbiculus specimens shared 717 unique OTUs. The 13 soritid specimens shared 26 OTUs, 23 of which represented Proteobacteria, predominantly of the bacterial family Rhodobacteraceae. The fourth foraminiferal species, Amphistegina gibbosa (Order Rotaliida) hosts diatom endosymbionts. Bacterial DNA extraction was attempted on 16 Am. gibbosa, including both normal-appearing and partly-bleached specimens. Only six OTUs, four of which represented Proteobacteria, were found in all eight specimens successfully sequenced. The partly bleached specimens shared nearly twice as many unique microbial OTUs (32) as the normal-appearing specimens (19). All Am. gibbosa specimens shared only four microbial OTUs with the soritid species, three of which may have been contaminants, indicating minimal commonality between the microbiomes of Am. gibbosa and the soritid taxa.

scholarly journals Field blood preservation and DNA extraction from wild mammals: methods and key factors for biodiversity studies

2021 ◽  
Vol 24 (1) ◽  
Author(s):  
Juan D. Carvajal-Agudelo ◽  
M. Paula Trujillo-Betancur ◽  
Daniela Velásquez-Guarín ◽  
Hector E. Ramírez-Chaves ◽  
Jorge E. Pérez-Cárdenas ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Guanidine Hydrochloride ◽  
Isoamyl Alcohol ◽  
Sample Collection ◽  
Total Blood ◽  
Blood Samples ◽  
Wild Mammals ◽  
Blood Preservation ◽  
Dna Preservation ◽  
Commercial Kits

Studies on public health and wild mammal biodiversity include a genetic component. For blood samples, there must be optimal sample collection conditions since these can affect DNA preservation and extraction. This study evaluated the use of liquid and dry DNA preservation methods and commercial and non-commercial DNA extraction methods on field-collected blood samples. For this, 264 total blood samples were collected from wild mammals. A first group of samples was preserved in guanidine hydrochloride (GuHCl) and DNA was extracted using six commercial kits:  Bioline, Norgen, Invitrogen, Promega, and Qiagen, in addition to phenol-chloroform isoamyl alcohol (PC) and guanidine thiocyanate (GIT). Another group of samples was preserved in Whatman® FTA® cards and DNA was extracted with PC and GIT. The extractions with GIT and PC showed the highest values (ng/µL) and variation in DNA concentration, while the commercial kit showed low variation. Sample preservation in Whatman® FTA® cards provided low variation and quantity of the extracted DNA compared with the use of GuHCl. Concerning DNA quality, the commercial kits yielded higher purity, while GIT and PC-based protocols provided highly variable results. Furthermore, the use of GIT and PC yielded a higher amount of DNA, yet, of variable quality. Overall, extraction based on commercial kits and Whatman® FTA® preservation allowed obtaining more standardized DNA qualities and quantities.

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