scholarly journals The Utilization of Linear Polylysine Coupled with Mechanic Forces to Extract Microbial DNA from Different Matrices

2020 ◽  
Vol 8 (12) ◽  
pp. 1901
Author(s):  
Celia François ◽  
Celia Martinez ◽  
Clement Faye ◽  
Nathalie Pansu ◽  
Catherine Dunyach-Remy ◽  
...  
Keyword(s):  
Cation Exchange Resin ◽  
Dna Recovery ◽  
Complex Samples ◽  
Pcr Inhibitors ◽  
Beverage Samples ◽  
Molecular Approaches ◽  
New Process ◽  
Polymerase Chain ◽  
Low Levels ◽  
Microbial Dna

Molecular approaches are powerful tools that are used for medical or environmental diagnoses. However, the main limitations of such a tools are that they extract low levels of DNA and they do not remove the inhibitors of polymerase chain reaction (PCR). Although the use of polycation to complex and purify DNA has been described in the literature, elution often requires a high ionic strength or pH levels not compatible with molecular analyses. In this paper, we described a new process that is based on the complexation of DNA with linear polylysine, followed by capturing the complex by a cation exchange resin. The originality of the process consisted of using mechanic force to elute DNA from the complex. The extraction method showed several advantages when compared to existing methods, such as being compatible with pH levels that range from 5 to 11, as well as high levels of DNA recovery and elimination of PCR inhibitors from complex samples. This method was successfully applied to different types of samples, such as environmental samples, beverage samples, and medical samples. Furthermore, it was proven to be a good solution for removing PCR inhibitors and assuring good DNA recovery yield.

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

scholarly journals Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

2017 ◽  
Vol 69 (6) ◽  
pp. 1443-1450 ◽  
Author(s):  
M.P. Campos ◽  
M.F. Madeira ◽  
D.A. Silva ◽  
M.S. Solcà ◽  
O.M. Espíndola ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Healthy Skin ◽  
Sample Collection ◽  
Chain Reaction ◽  
Dna Recovery ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Polymerase Chain ◽  
Commercial Kits

ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

Kato–Katz thick smears as a DNA source of soil-transmitted helminths

2018 ◽  
Vol 94 ◽  
Author(s):  
K.J.L. Monteiro ◽  
D.A. Calegar ◽  
F.A. Carvalho-Costa ◽  
L.H. Jaeger ◽  
Keyword(s):  
Evolutionary Genetics ◽  
Large Collection ◽  
Chain Reaction ◽  
Rural Regions ◽  
Dna Recovery ◽  
Polymerase Chain ◽  
N 93 ◽  
Initial Processing ◽  
Specific Species

AbstractDespite the reduction in the prevalence of soil-transmitted helminthiases in many regions of the world, morbidity rates remain high in some rural regions. The Kato–Katz technique is a simple, inexpensive and field-applicable tool commonly used for the diagnosis and worm-burden characterization of these infections. Molecular studies have revolutionized our understanding of the epidemiology and evolutionary genetics of parasites. In this study we recovered helminthic DNA from Kato–Katz slides (n = 93) prepared in 2011 in the Brazilian Amazon. We achieved DNA recovery by polymerase chain reaction (PCR) in 84% of cases for Ascaris sp. and 75% of cases for hookworms. The sequencing confirmed the specific species of the amplicons. The slides stored for a few years could be analysed using this methodology, allowing access to DNA from a large collection of samples. We must consider the Kato–Katz thick smears as a source of helminth DNA. This can significantly reduce logistical difficulties in the field in terms of obtaining, preserving, transporting and initial processing of samples.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

scholarly journals Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Eva Malatinkova ◽  
Ward De Spiegelaere ◽  
Pawel Bonczkowski ◽  
Maja Kiselinova ◽  
Karen Vervisch ◽  
...  
Keyword(s):  
Antiretroviral Therapy ◽  
Treatment Initiation ◽  
Cross Sectional Study ◽  
Functional Cure ◽  
Cross Sectional ◽  
Polymerase Chain ◽  
Low Levels ◽  
Rectal Biopsies ◽  
Hiv 1

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure.

scholarly journals Continuous Microfluidic Purification of DNA Using Magnetophoresis

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 187
Author(s):  
Ying Xu ◽  
Zhen Zhang ◽  
Zhen Su ◽  
Xiaoxiang Zhou ◽  
Xiaoming Han ◽  
...  
Keyword(s):  
Dna Isolation ◽  
Fluid Velocity ◽  
Microfluidic System ◽  
Recovery Efficiency ◽  
Ndfeb Magnet ◽  
Chain Reaction ◽  
Dna Recovery ◽  
Fluid Velocities ◽  
Polymerase Chain ◽  
The Relationship

Automatic microfluidic purification of nucleic acid is predictable to reduce the input of original samples and improve the throughput of library preparation for sequencing. Here, we propose a novel microfluidic system using an external NdFeB magnet to isolate DNA from the polymerase chain reaction (PCR) mixture. The DNA was purified and isolated when the DNA-carrying beads transported to the interface of multi-laminar flow under the influence of magnetic field. Prior to the DNA recovery experiments, COMSOL simulations were carried out to study the relationship between trajectory of beads and magnet positions as well as fluid velocities. Afterwards, the experiments to study the influence of varying velocities and input of samples on the DNA recovery were conducted. Compared to experimental results, the relative error of the final position of beads is less than 10%. The recovery efficiency decreases with increase of input or fluid velocity, and the maximum DNA recovery efficiency is 98.4% with input of l00 ng DNA at fluid velocity of 1.373 mm/s. The results show that simulations significantly reduce the time for parameter adjustment in experiments. In addition, this platform uses a basic two-layer chip to realize automatic DNA isolation without any other liquid switch value or magnet controller.

scholarly journals Total Versus Free Placental Growth Factor Levels in the Pathogenesis of Preeclampsia

Hypertension ◽  
2020 ◽  
Vol 76 (3) ◽  
pp. 875-883
Author(s):  
Edouard Lecarpentier ◽  
Zsuzsanna K. Zsengellér ◽  
Saira Salahuddin ◽  
Ambart E. Covarrubias ◽  
Agnes Lo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ex Vivo ◽  
Quantitative Polymerase Chain Reaction ◽  
Placental Growth Factor ◽  
Protein Levels ◽  
Key Characteristics ◽  
Human Blood Samples ◽  
Polymerase Chain ◽  
Low Levels ◽  
Independent Cohort

Elevated circulating sFLT-1 (soluble fms-like tyrosine kinase) and low levels of its ligand, PlGF (placental growth factor), are key characteristics of preeclampsia. However, it is unclear if the low levels of plasma PlGF noted during preeclampsia are due to decreased placental production of PlGF or due to binding of PlGF by increased circulating sFLT-1. Here, we describe a biochemical procedure to dissociate PlGF-sFLT-1 complex ex vivo and when used in conjunction with an immunoassay platform, demonstrate a method to measure total and free PlGF in human blood samples. Using this method, we noted that plasma free PlGF levels were significantly lower in preeclampsia (N=22) than in nonhypertensive controls (N=24; mean, 314 versus 686 pg/mL, P <0.05), but total PlGF levels were not different (mean, 822 versus 800 pg/mL, P =0.49). In contrast, total sFLT-1 levels were significantly higher in preeclampsia than in nonhypertensive controls (mean, 16 957 versus 3029 pg/mL, P <0.01) and sFLT-1 levels correlated with bound PlGF levels (bound PlGF=total PlGF−free PlGF) in these samples ( r 2 =0.68). We confirmed these findings in an independent cohort of subjects (N=49). Furthermore, we did not detect any difference in PlGF mRNA by quantitative polymerase chain reaction or in PlGF protein expression by immunohistochemistry in preeclamptic placentas when compared with nonhypertensive controls. In contrast, sFLT-1 mRNA and protein levels were upregulated in placentas from women with preeclampsia. Taken together with prior studies, our results provide evidence that decrease in circulating PlGF noted during preeclampsia is largely mediated by excess circulating sFLT-1.

A comprehensive search for microbial DNA and RNA in sarcoidosis tissue samples by quantitative polymerase chain reaction

2017 ◽  
Vol 49 (8) ◽  
pp. 625-627
Author(s):  
Fumio Terasaki ◽  
Hitomi Fukumoto ◽  
Ryo Kawata ◽  
Yoshinobu Hirose ◽  
Shu-ichi Fujita ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Dna And Rna ◽  
Comprehensive Search ◽  
Polymerase Chain ◽  
Microbial Dna
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