scholarly journals Performance comparison of different microbial DNA extraction methods on bird feces

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xian Hou ◽  
Shengkai Pan ◽  
Zhenzhen Lin ◽  
Jiliang Xu ◽  
Xiangjiang Zhan
Keyword(s):  
Dna Extraction ◽  
Relative Abundance ◽  
Alpha Diversity ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Dna Integrity ◽  
Microbial Composition ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Microbial Dna

Abstract Background As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

scholarly journals Impact of bead-beating intensity on microbiome recovery in mouse and human stool: Optimization of DNA extraction

2020 ◽  
Author(s):  
Bo Zhang ◽  
Matthew Brock ◽  
Carlos Arana ◽  
Chaitanya Dende ◽  
Lora Hooper ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Dna Integrity ◽  
Sequencing Data ◽  
16S Sequencing ◽  
Bead Beating ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
The Impact ◽  
Human Stool

AbstractDNA extraction methods play an important role in the acquisition of accurate and reproducible 16S sequencing data in microbiome studies. In this study, we assessed the impact of bead-beating intensity during DNA extraction on microbiome recovery in mouse and human stool. We observed a higher DNA yield, better DNA integrity, higher Shannon’s entropy and Simpson’s index in samples beaten for 4 and 9 minutes as compared to unbeaten samples. 16S sequencing data showed that bead beating has a statistically-significant (p<0.05) impact on the recovery of many clinically relevant microbes that live in the mouse and human gut, including Bifidobacterium, Sutterella and Veillonella. It was observed that 4 minutes of bead beating promotes recovery of about 70% of OTUs in mouse and human stool, while the remaining 30% requires longer bead beating. In conclusion, our study indicates adjustments in bead beating treatment based on the composition of the specimen and the targeted bacteria.

scholarly journals Heat Inactivation of Coccidioides posadasii and Coccidioides immitis for Use in Lower Biosafety Containment

2019 ◽  
Vol 24 (3) ◽  
pp. 123-128 ◽  
Author(s):  
Heather L. Mead ◽  
Austin V. Blackmon ◽  
Amy J. Vogler ◽  
Bridget M. Barker
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Extraction Methods ◽  
Heat Inactivation ◽  
Dna Integrity ◽  
Coccidioides Immitis ◽  
Coccidioides Posadasii ◽  
Dna Yield ◽  
Level 3 ◽  
Ideal Method

Introduction: The difficulty involved in obtaining sufficient intact genomic deoxyribonucleic acid (DNA) from Coccidioides spp for downstream applications using published protocols prompted the exploration of inactivating mycelia and arthroconidia using heat under biosafety level 3 containment. This was followed by optimizing DNA extraction from mycelia using various methods at lower containment. Methods: Various exposure times and temperatures were examined to identify an effective heat inactivation procedure for arthroconidia and mycelia from both C immitis and C posadasii. Heat inactivation of mycelia was followed by DNA extraction using 2 commercially available kits, as well as a phenol:chloroform-based extraction procedure to determine DNA integrity and quantity among extraction methods using both live and heat-inactivated mycelia. Results: Ten-minute and 30-minute exposure times at 80°C were sufficient to inactivate Coccidioides spp arthroconidia and mycelia, respectively. DNA yield between live versus heat-inactivated mycelia was similar for each extraction procedure. However, DNA obtained using phenol:chloroform was of higher quantity and integrity compared with DNA obtained using the commercially available kits, which was highly fragmented. Conclusion: The ability to heat-inactivate Coccidioides cultures for processing at a lower level of containment greatly increased the efficiency of DNA extractions. Therefore, this is an ideal method for obtaining Coccidioides spp DNA and inactivated arthroconidia.

scholarly journals Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262355
Author(s):  
Elinor Shvartsman ◽  
Meika E. I. Richmond ◽  
John J. Schellenberg ◽  
Alana Lamont ◽  
Catia Perciani ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Female Genital Tract ◽  
Alpha Diversity ◽  
Sample Type ◽  
Microbial Composition ◽  
Dna Yield ◽  
Microbial Profiling ◽  
Pcr Product ◽  
Purification Methods ◽  
Pre Treatment

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

scholarly journals At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9109
Author(s):  
Marta Saługa
Keyword(s):  
Dna Extraction ◽  
Extreme Environments ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Herbarium Specimens ◽  
Polar Regions ◽  
Moss Species ◽  
Dna Yield ◽  
Herbarium Collections ◽  
Dna Extraction Methods

Background Research focused on extreme environments is often associated with difficulties in obtaining fresh plant material. Herbaria may provide great support as they house large collections of specimens from different parts of the world. Accordingly, there is also a growing interest in methods using herbarium specimens in molecular studies. Much of the literature on herbarium DNA is aimed to improve extraction and PCR amplification and is focused mostly on vascular plants. Here, I provide a brief study of DNA extraction efficiency from moss herbarium specimens, emphasizing the importance of herbaria as an invaluable source of material from hard-to-access geographical areas, such as the Antarctic region. Methods The presented study is based on herbarium collections of 25 moss species collected in the austral polar regions between 1979 and 2013. The majority of samples were obtained using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The remaining, smaller part was extracted using an adapted CTAB-based approach. The performance of DNA extraction methods in terms of PCR amplification success was measured by testing several DNA fragments of various size. Furthermore, in order to estimate of DNA fragmentation level, an automated on-chip electrophoresis system was used. Results Results reveal that DNA purity and the length of the target genetic region are the fundamental agents which drive the successful PCR reaction. Conversely, the DNA yield and specimen age seem to be less relevant. With this study, I present also an optimized CTAB-based approach which may effectively suppress inhibitors in the herbarium DNA. This method can be considered a cheaper alternative to column-based technology, particularly useful for dealing with a large number of samples. Results of this study confirmed previous reports and contribute to filling the existing gap in molecular analyses which involve the use of herbarium collections of mosses.

scholarly journals Correction to: Performance comparison of different microbial DNA extraction methods on bird feces

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xian Hou ◽  
Shengkai Pan ◽  
Zhenzhen Lin ◽  
Jiliang Xu ◽  
Xiangjiang Zhan
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
Performance Comparison ◽  
Dna Extraction Methods ◽  
Microbial Dna ◽  
Bird Feces

An amendment to this paper has been published and can be accessed via the original article.

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

2010 ◽  
Vol 81 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Anne Salonen ◽  
Janne Nikkilä ◽  
Jonna Jalanka-Tuovinen ◽  
Outi Immonen ◽  
Mirjana Rajilić-Stojanović ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Dna Extraction ◽  
Cell Lysis ◽  
Extraction Methods ◽  
Fecal Dna ◽  
Dna Extraction Methods

scholarly journals Analysis of automatically extracted white blood cell DNA yield

2020 ◽  
pp. 40-50
Author(s):  
М.В. Олькова ◽  
Е.В. Балановская ◽  
Л.С. Бычковская ◽  
О.П. Балановский
Keyword(s):  
Blood Cell ◽  
Dna Extraction ◽  
White Blood Cell ◽  
Regression Models ◽  
Extraction Methods ◽  
Reference Points ◽  
Linear Regression Models ◽  
Dna Concentration ◽  
Dna Yield ◽  
Dna Extraction Methods

Все более широкое применение поточных методов экстракции ДНК влечет за собой необходимость стандартизации и проверки качества ее выделения. Мы провели детальное количественное изучение процесса выделения и определили стандартизирующие опорные точки для одного из наиболее производительных методов - выделения на магнитных частицах с использованием автоматической станции QIAsymphony SP. Показано, что концентрация ДНК в индивидуальном образце в основном определяется содержанием лейкоцитов в исходном образце крови (корреляция около 0,9). Концентрация ДНК также зависит от метода измерения. Это позволило нам построить линейные регрессионные модели и вывести формулы, точно прогнозирующие концентрацию ДНК в образце для случаев применения двух широко распространенных методов - спектрофотометрического (Nanodrop) и флуоресцентного (Qubit). Обнаружено, что последняя модель Nanodrop OneC, благодаря встроенному алгоритму идентификации примесей и корректировки концентрации, дает более точную оценку концентрации, чем Qubit 4.0. Для быстрого, но приблизительного прогноза концентрации ДНК вместо регрессионных моделей могут применяться стандарты, рассчитанные нами для референсных значений содержания лейкоцитов. The continuing development of mass DNA extraction methods entails the need to set standardisation and quality verification reference points. A study has been conducted and standards set for one of the many methods of DNA extraction - the automated мagnetic beads-based extraction using QIAsymphony SP station. It was shown that the concentration of DNA in an individual sample is mainly determined by the leukocyte content in the initial blood sample (correlation of about 0.9). DNA concentration also depends on the measurement method. It allowed us to build linear regression models and derive formulae that accurately predict the concentration of DNA in the sample for the use of two widely used methods - spectrophotometric (Nanodrop) and fluorescence (Qubit). It was found that the latest Nanodrop OneC model, thanks to a built-in algorithm for identifying impurities and adjusting the concentration, provides an even more accurate concentration estimate than Qubit 4.0. For a quick but rough forecast of DNA concentration, instead of regression models, standards calculated by us for reference values of the white blood cell count can be used.

Quantifying technical confounders in microbiome studies

2020 ◽  
Author(s):  
Theda U P Bartolomaeus ◽  
Till Birkner ◽  
Hendrik Bartolomaeus ◽  
Ulrike Löber ◽  
Ellen G Avery ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Human Microbiome ◽  
Alpha Diversity ◽  
Extraction Methods ◽  
Intestinal Microbiome ◽  
Interindividual Differences ◽  
Microbiome Composition ◽  
Technical Developments ◽  
Dna Extraction Methods ◽  
The Impact

Abstract Aims Recent technical developments have allowed the study of the human microbiome to accelerate at an unprecedented pace. Methodological differences may have considerable impact on the results obtained. Thus, we investigated how different storage, isolation, and DNA extraction methods can influence the characterization of the intestinal microbiome, compared to the impact of true biological signals such as intraindividual variability, nutrition, health, and demographics. Methods and results An observative cohort study in 27 healthy subjects was performed. Participants were instructed to collect stool samples twice spaced by a week, using six different methods (naive and Zymo DNA/RNA Shield on dry ice, OMNIgene GUT, RNALater, 95% ethanol, Zymo DNA/RNA Shield at room temperature). DNA extraction from all samples was performed comparatively using QIAamp Power Fecal and ZymoBIOMICS DNA Kits. 16S rRNA sequencing of the gut microbiota as well as qPCRs were performed on the isolated DNA. Metrics included alpha diversity as well as multivariate and univariate comparisons of samples, controlling for covariate patterns computationally. Interindividual differences explained 7.4% of overall microbiome variability, whereas the choice of DNA extraction method explained a further 5.7%. At phylum level, the tested kits differed in their recovery of Gram-positive bacteria, which is reflected in a significantly skewed enterotype distribution. Conclusion DNA extraction methods had the highest impact on observed microbiome variability, and were comparable to interindividual differences, thus may spuriously mimic the microbiome signatures of various health and nutrition factors. Conversely, collection methods had a relatively small influence on microbiome composition. The present study provides necessary insight into the technical variables which can lead to divergent results from seemingly similar study designs. We anticipate that these results will contribute to future efforts towards standardization of microbiome quantification procedures in clinical research.

scholarly journals Endocervical swabs transported in first void urine as combined specimens in the detection of Mycoplasma genitalium by real-time PCR

2009 ◽  
Vol 58 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Andreas Edberg ◽  
Fredrik Aronsson ◽  
Eva Johansson ◽  
Elisabeth Wikander ◽  
Thomas Ahlqvist ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Mycoplasma Genitalium ◽  
Extraction Methods ◽  
Pcr Inhibition ◽  
Dna Yield ◽  
Extraction Study ◽  
Dna Extraction Methods ◽  
Swab Specimen

The aim of this study was to determine whether a patient's endocervical swab specimen can be transported in first void urine (FVU) as combined specimens for the detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods for observation of possible PCR inhibition. Three specimens, one endocervical swab specimen transported in 2-SP medium, one endocervical swab specimen transported in FVU and a FVU specimen, were collected from 329 women. All sample types underwent manual DNA extraction whereas in the DNA extraction study, 329 endocervical swab specimens transported in FVU were subjected to both manual Chelex and automated BioRobot M48 DNA extraction. A total of 100 endocervical swab specimens transported in FVU from patients PCR-negative for M. genitalium in the study were used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. The endocervical swab specimens transported in 2-SP medium and transported in FVU were positive for M. genitalium in 17/25 (68 %) and 24/25 (96 %) women, respectively. The FVU specimens alone were positive for M. genitalium in 22/25 (88 %) women. In the DNA extraction study, M. genitalium DNA was detected in 24/329 (7.3 %) and 28/329 (8.5 %) of endocervical swab specimens transported in FVU subjected to manual Chelex extraction and automated BioRobot M48 extraction, respectively. Partial PCR inhibition was detected in 6 % of samples subjected to manual Chelex extraction whereas no inhibition was detected with the automated BioRobot M48 extraction. Thus endocervical swab specimens transported in FVU demonstrate higher sensitivity than FVU specimens only and have considerably increased sensitivity compared with endocervical swab specimens transported in 2-SP medium for detection of M. genitalium DNA. Moreover, automated BioRobot M48 extraction was shown to be superior to a crude manual Chelex extraction, leaving no PCR inhibition and giving a slightly higher DNA yield and/or better sensitivity.

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