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 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.

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 The Viable Microbiome of Human Milk Differs from the Metataxonomic Profile

Nutrients ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 4445
Author(s):  
Lisa F. Stinson ◽  
Michelle L. Trevenen ◽  
Donna T. Geddes
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Human Milk ◽  
Dna Content ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Dna ◽  
Cutibacterium Acnes ◽  
Rrna Gene Sequencing

Bacteria in human milk contribute to the establishment of the infant gut microbiome. As such, numerous studies have characterized the human milk microbiome using DNA sequencing technologies, particularly 16S rRNA gene sequencing. However, such methods are not able to differentiate between DNA from viable and non-viable bacteria. The extent to which bacterial DNA detected in human milk represents living, biologically active cells is therefore unclear. Here, we characterized both the viable bacterial content and the total bacterial DNA content (derived from viable and non-viable cells) of fresh human milk (n = 10). In order to differentiate the living from the dead, a combination of propidium monoazide (PMA) and full-length 16S rRNA gene sequencing was used. Our results demonstrate that the majority of OTUs recovered from fresh human milk samples (67.3%) reflected DNA from non-viable organisms. PMA-treated samples differed significantly in their bacterial composition compared to untreated samples (PERMANOVA p < 0.0001). Additionally, an OTU mapping to Cutibacterium acnes had a significantly higher relative abundance in PMA-treated (viable) samples. These results demonstrate that the total bacterial DNA content of human milk is not representative of the viable human milk microbiome. Our findings raise questions about the validity of conclusions drawn from previous studies in which viability testing was not used, and have broad implications for the design of future work in this field.

scholarly journals TAMI-40. TUMOR MICROBIOME AND GLIOBLASTOMA (GBM)

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii221-ii222
Author(s):  
Shlomit Yust-Katz ◽  
Elinor Gigi ◽  
Deborah Rosenberg ◽  
Andrew A Kanner ◽  
Yoseph Laviv ◽  
...  
Keyword(s):  
16S Rrna ◽  
Lipoteichoic Acid ◽  
Study Cohort ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Bacterial Dna ◽  
Dna Recovery ◽  
Bacterial Rna ◽  
Human Gbm ◽  
Negative Controls

Abstract Bacteria have been found to play major roles in many physiologic/disease processes including cancer. The presence of bacteria within brain tumors has never been explored. The aim of this study was to examine the microbiome of Glioblastoma. A cohort of 40 glioblastoma samples (FFPE), from two medical centers served for DNA extraction using a specialized extraction protocol that includes a bead beating step to ensure complete bacterial DNA recovery. A set of negative controls was introduced at different steps of the assay to identify and monitor contaminating bacterial DNA. We measured the levels of bacterial DNA in the samples using a RT-qPCR assay, amplifying the bacterial 16S rRNA gene and detected bacterial DNA in over 40% of the samples. To characterize the bacterial taxa that are present in GBM tumors, we applied 16S DNA sequencing on the samples. After implementing a stringent set of filters on the sequencing data, eliminating contaminating signal, we detected a total of 22 bacterial taxa in GBM tumors. To visualize bacteria in GBM tissues and learn about their localization within the tissue we used immunohistochemistry staining with anti-lipopolysaccharide (LPS) and anti-lipoteichoic acid (LTA) antibodies detecting gram negative and gram positive bacteria (correspondingly). Bacteria were also visualized by staining bacterial RNA using a 16S rRNA in situ hybridization assay. Staining of a human GBM tissue microarray (TMA) containing 32 cases of GBM showed that the majority of cases stained positive for LPS and ~40% were positive for 16S rRNA staining. Bacterial LPS and 16S rRNA were localized mainly inside the tumor cells. Our study demonstrates, for the first time, that bacteria or bacterial components are present in human Glioblastoma tumors. We are currently expanding our study cohort in order to better define the bacteria found within glioblastoma samples and assess their possible effects

scholarly journals The microbiota of the surface, dermis and subcutaneous tissue of dog skin

2020 ◽  
Author(s):  
Rocío García-Fonticoba ◽  
Lluis Ferrer ◽  
Olga Francino ◽  
Anna Cusco
Keyword(s):  
Subcutaneous Tissue ◽  
Dna Amplification ◽  
Skin Surface ◽  
Molecular Techniques ◽  
The Other ◽  
Rrna Gene ◽  
Culture Methods ◽  
Bacterial Dna ◽  
Microbiological Culture ◽  
Two Samples

Abstract Background. Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this finding could be clinically relevant. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of dogs without cutaneous disease using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS).Results. Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA amplification and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in five out of the seven skin surface swab samples with aerobic microbiological culture methods, while no growth was obtained from the other two samples. Although some DNA could be amplified from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks.Conclusion. When investigated with aerobic microbiological culture methods, the dermis and subcutaneous tissue of dogs are sterile. NGS techniques lead to the detection of some bacterial DNA, similar to the signal detected in blanks, which does not support the presence of a microbiota in dermis or subcutaneous tissue.

scholarly journals A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  
Keyword(s):  
Dna Extraction ◽  
Foodborne Pathogens ◽  
Extraction Method ◽  
Pathogenic Bacteria ◽  
Low Cost ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Bacterial Dna ◽  
Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

scholarly journals Biases during DNA extraction affect characterization of the microbiota associated with larvae of the Pacific white shrimp,Litopenaeus vannamei

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5257 ◽  
Author(s):  
Ming Xue ◽  
Liyou Wu ◽  
Yaoyao He ◽  
Huafang Liang ◽  
Chongqing Wen
Keyword(s):  
Dna Extraction ◽  
Litopenaeus Vannamei ◽  
Penaeid Shrimp ◽  
Illumina Miseq ◽  
Pacific White Shrimp ◽  
White Shrimp ◽  
Dna Recovery ◽  
The Pacific ◽  
Α Diversity

For in-depth characterization of the microbiota associated with shrimp larvae, careful selection of DNA isolation procedure is paramount for avoiding biases introduced in community profiling. Four E.Z.N.A.™ DNA extraction kits, i.e., Bacterial, Mollusc, Stool, and Tissue DNA Kits, abbreviated as Ba, Mo, St, and Ti, respectively, were initially evaluated with zoea 2 (Z2) larvae of the Pacific white shrimp (Litopenaeus vannamei) by 16S amplicon sequencing on a Illumina MiSeq platform. Further characterization of additional larval samples, specifically nauplii 5 (N5), mysis 1 (M1), and postlarvae 1 (P1), was performed with Ba and St kits to examine the changing microbiota profile during shrimp hatchery period. The results from the Z2 samples showed that DNA yields from the four kits varied significantly (P< 0.05), whereas no significant differences were detected in the α-diversity metrics of the microbiota. By contrast, the St kit, with the lowest DNA yield and quality, successfully recovered DNA from Gram-positive and gut-associated bacterial groups, whereas the Ba kit, which showed maximal microbiota similarity with the Mo kit, manifested the best reproducibility. Notably, significant differences were observed in relative abundances of most dominant taxa when comparing results from the Ba and St kits on Z2, M1, and P1 samples. In addition, the bacterial community identified shifted markedly with larval development regardless of the DNA extraction kits. The DNA recovery biases arising from the larval microbiota could be due to different protocols for cell lysis and purification. Therefore, combined application of different DNA extraction methods may facilitate identification of some biologically important groups owing to their complementary effects. This approach should receive adequate attention for a thorough understanding of the larvae-associated microbiota of the penaeid shrimp.

Design of PCR Primers for the mcrA Genes Detection of Methanogen Communities

2011 ◽  
Vol 135-136 ◽  
pp. 408-413 ◽  
Author(s):  
Nguyen Ngoc Tuan ◽  
Shir Ly Huang
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Biogas Production ◽  
Pcr Primers ◽  
Molecular Techniques ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Mcra Gene ◽  
Catabolic Genes ◽  
Culture Independent ◽  
Gradient Gel Electrophoresis

Methanogens play an important role to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent green house gases, during organic matter degradation in anaerobic environments. Therefore, it is necessary to better understand microorganisms that produce natural gas. Indeed, methanogens are difficult to perform through culture based methods. In addition, the culture independent methods using the 16S rRNA gene also revealed some disadvantages. For these reasons, the culture independent molecular techniques using the specific catabolic genes such as methyl coenzyme M reductase (MCR) were studied. In this study, a primer set which can amplify specific fragments from a wide variety of mcrA gene was designed based on the homologous regions of 100 mcrA genes listed in the GenBank. PCR with the mcrA primers amplified DNA fragments of the expected size from all the six samples which obtained from biogas production reactors. In addition, denaturing gradient gel electrophoresis PCR analysis using our designed primers also revealed the diversity of mcrA gene in each sample. These results revealed that our primers were successfully to detect the mcrA genes and it is also helpful to know the diversity of mcrA genes in methanogen communities.

scholarly journals Culture-Dependent and -Independent Characterization of Microbial Communities Associated with a Shallow Submarine Hydrothermal System Occurring within a Coral Reef off Taketomi Island, Japan

2007 ◽  
Vol 73 (23) ◽  
pp. 7642-7656 ◽  
Author(s):  
Hisako Hirayama ◽  
Michinari Sunamura ◽  
Ken Takai ◽  
Takuro Nunoura ◽  
Takuro Noguchi ◽  
...  
Keyword(s):  
Coral Reef ◽  
Microbial Communities ◽  
Hydrothermal System ◽  
Hydrothermal Activity ◽  
Molecular Techniques ◽  
Maximum Temperature ◽  
Rrna Gene ◽  
Type I ◽  
Optimum Growth ◽  
Culture Independent

ABSTRACT Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, ∼23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52°C) contained 175 μM H2S and gas bubbles mainly composed of CH4 (69%) and N2 (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37°C, and another novel type I methanotroph exhibited optimum growth at 45°C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.

scholarly journals The microbiota of the surface, dermis and subcutaneous tissue of dog skin

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Rocío García-Fonticoba ◽  
Lluís Ferrer ◽  
Olga Francino ◽  
Anna Cuscó
Keyword(s):  
Subcutaneous Tissue ◽  
Dna Amplification ◽  
Skin Surface ◽  
Molecular Techniques ◽  
The Other ◽  
Rrna Gene ◽  
Culture Methods ◽  
Bacterial Dna ◽  
Microbiological Culture ◽  
Two Samples

Abstract Background Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this finding could be clinically relevant. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of dogs without cutaneous disease using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS). Results Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA amplification and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in five out of the seven skin surface swab samples with aerobic microbiological culture methods, while no growth was obtained from the other two samples. Although some DNA could be amplified from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks. Conclusion When investigated with aerobic microbiological culture methods, the dermis and subcutaneous tissue of dogs are sterile. NGS techniques lead to the detection of some bacterial DNA, similar to the signal detected in blanks, which does not support the presence of a microbiota in dermis or subcutaneous tissue.

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