scholarly journals Preliminary Evidence of a Molecular Detection Method to Analyze Bacterial DNA as a Quality Indicator in Cosmetics

Cosmetics ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 54
Author(s):  
Luca Michelutti ◽  
Michela Bulfoni ◽  
Veronica Bolzon ◽  
Emanuele Nencioni
Keyword(s):  
Bacterial Load ◽  
Extraction Methods ◽  
Preliminary Evidence ◽  
Rapid Identification ◽  
Plate Count ◽  
Cell Recovery ◽  
Easy Method ◽  
Dna Yield ◽  
Microorganism Identification ◽  
Dna Extraction Methods

Cosmetics are a category of widely consumed and distributed products, and their manufacture is always subject to specific guidelines. Quality Control (QC) tests provide information supporting the absence of injurious organisms and regarding the microbiological stability of cosmetics. The microbiological risk analysis is typically performed using the plate count method, which is a time-consuming and operator-dependent approach. Molecular technologies allow a deeper and more sensitive testing than traditional cultures. The demand for rapid and sensitive methods is recently increasing. The aim of our study was to compare different DNA extraction methods in order to detect and quantify bacterial load in cosmetics using a qPCR system. Known numbers of microorganisms were spiked into six different cosmetics to simulate contaminated samples. DNA was extracted with seven extraction kits and then quantified by real-time qPCR. Results revealed differences in terms of cell recovery, DNA yield, and quality. The bead-beating approaches were the most suitable in our molecular workflow and lead to good quality DNA for analysis by qPCR within four hours. Combined with mechanical extraction, qPCR may represent an efficient and easy method for microorganism identification in cosmetics, and can be automated. This approach also is also applicable for the detection of probiotics used as beneficial biological components in cosmetic products. The results of our molecular method provided preliminary evidences for the rapid identification of cells (10–100) and nucleic acids in complex preparations employed for human health, in compliance with regulatory limits. The suggested methodology is easy, fast, and sensitive. Its scalability allows serial microbiological evaluation at every manufacturing step.

scholarly journals Improving sustainable use of genetic resources in biodiversity archives

2019 ◽  
Author(s):  
E J Tuschhoff ◽  
Carl R Hutter ◽  
Richard E Glor
Keyword(s):  
Genetic Resources ◽  
Tissue Sample ◽  
Sustainable Use ◽  
Extraction Methods ◽  
Original Material ◽  
Tissue Mass ◽  
Tissue Samples ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Sample Age

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are frequently destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 mg and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

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 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 Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf samples

2021 ◽  
Author(s):  
Veronica Roman-reyna ◽  
Enora Dupas ◽  
Sophie Cesbron ◽  
Guido Marchi ◽  
Sara Campigli ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Xylella Fastidiosa ◽  
Management Strategies ◽  
Infected Plant ◽  
The United States ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Metagenomic Sequencing ◽  
Primary Control ◽  
Dna Extraction Methods

Xylella fastidiosa (Xf) is a globally distributed plant pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to strain level in single and mixed infected plant samples at concentrations as low as one picogram of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (Cq > 35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. We hope this pipeline can be used for early detection of Xf and incorporated as a tool to inform disease management strategies.

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 Improving sustainable use of genetic resources in biodiversity archives

2019 ◽  
Author(s):  
E J Tuschhoff ◽  
Carl R Hutter ◽  
Richard E Glor
Keyword(s):  
Genetic Resources ◽  
Tissue Sample ◽  
Sustainable Use ◽  
Extraction Methods ◽  
Original Material ◽  
Tissue Mass ◽  
Tissue Samples ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Sample Age

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are frequently destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 mg and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

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.

scholarly journals Improving sustainable use of genetic resources in biodiversity archives

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8369
Author(s):  
E. J. Tuschhoff ◽  
Carl R. Hutter ◽  
Richard E. Glor
Keyword(s):  
Genetic Resources ◽  
Tissue Sample ◽  
Sustainable Use ◽  
Extraction Methods ◽  
Original Material ◽  
Tissue Mass ◽  
Tissue Samples ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Sample Age

Tissue sample databases housed in biodiversity archives represent a vast trove of genetic resources, and these tissues are often destructively subsampled and provided to researchers for DNA extractions and subsequent sequencing. While obtaining a sufficient quantity of DNA for downstream applications is vital for these researchers, it is also important to preserve tissue resources for future use given that the original material is destructively and consumptively sampled with each use. It is therefore necessary to develop standardized tissue subsampling and loaning procedures to ensure that tissues are being used efficiently. In this study, we specifically focus on the efficiency of DNA extraction methods by using anuran liver and muscle tissues maintained at a biodiversity archive. We conducted a series of experiments to test whether current practices involving coarse visual assessments of tissue size are effective, how tissue mass correlates with DNA yield and concentration, and whether the amount of DNA recovered is correlated with sample age. We found that tissue samples between 2 and 8 mg resulted in the most efficient extractions, with tissues at the lower end of this range providing more DNA per unit mass and tissues at the higher end of this range providing more total DNA. Additionally, we found no correlation between tissue age and DNA yield. Because we find that even very small tissue subsamples tend to yield far more DNA than is required by researchers for modern sequencing applications (including whole genome shotgun sequencing), we recommend that biodiversity archives consider dramatically improving sustainable use of their archived material by providing researchers with set quantities of extracted DNA rather than with the subsampled tissues themselves.

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.

scholarly journals A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods

2020 ◽  
Vol 13 (2) ◽  
pp. 124-127
Author(s):  
Ishwar Prasad Dubey ◽  
R. K. Kumawat ◽  
I. P. Tripathi ◽  
Pankaj Shrivastava
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Forensic Dna ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Short Tandem

Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.

Sign in / Sign up

Export Citation Format

Share Document