scholarly journals To beat or not to beat a tick: Comparison of DNA extraction methods from ticks (Ixodes scapularis)

2015 ◽  
Author(s):  
Alyssa D Ammazzalorso ◽  
Christine P Zolnik ◽  
Thomas J Daniels ◽  
Sergios-Orestis Kolokotronis
Keyword(s):  
Dna Extraction ◽  
Disease Transmission ◽  
Dna Isolation ◽  
Ixodes Scapularis ◽  
Genetic Material ◽  
The United States ◽  
Qualitative Assessment ◽  
Single Individual ◽  
Cross Sectional ◽  
Dna Yield

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis – nymph and adult - that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification kit resulted in the highest DNA yields and the strongest, most consistent PCR amplification. We also found that physical disruption of the tick exoskeleton was most effective using cross-sectional cutting compared to any type of bead-beating matrices used. Storing ticks at -80°C resulted in considerably higher DNA yields than those from ticks stored in ethanol. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

scholarly journals To beat or not to beat a tick: Comparison of DNA extraction methods from ticks (Ixodes scapularis)

2015 ◽  
Author(s):  
Alyssa D Ammazzalorso ◽  
Christine P Zolnik ◽  
Thomas J Daniels ◽  
Sergios-Orestis Kolokotronis
Keyword(s):  
Dna Extraction ◽  
Disease Transmission ◽  
Dna Isolation ◽  
Ixodes Scapularis ◽  
Genetic Material ◽  
The United States ◽  
Qualitative Assessment ◽  
Single Individual ◽  
Cross Sectional ◽  
Dna Yield

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis – nymph and adult - that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification kit resulted in the highest DNA yields and the strongest, most consistent PCR amplification. We also found that physical disruption of the tick exoskeleton was most effective using cross-sectional cutting compared to any type of bead-beating matrices used. Storing ticks at -80°C resulted in considerably higher DNA yields than those from ticks stored in ethanol. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

2011 ◽  
Vol 57 (8) ◽  
pp. 623-628 ◽  
Author(s):  
Nagissa Mahmoudi ◽  
Greg F. Slater ◽  
Roberta R. Fulthorpe
Keyword(s):  
Dna Extraction ◽  
Contaminated Soils ◽  
Dna Isolation ◽  
Extraction Process ◽  
Organic Carbon Content ◽  
Soil Dna ◽  
Isolation And Purification ◽  
Dna Yield ◽  
Pcr Products ◽  
Eukaryotic Dna

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid–liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

Efficient DNA isolation from moroccan arar tree [Tetraclinis articulata (Vahl) Masters] leaves and optimization of the rapd-pcr molecular technique. Extraction efficace de l’ADN des feuilles du thuya de berberie (Tetraclinis articulata (Vahl) masters) et optimisation de la technique moléculaire rapd-pcr

2010 ◽  
Vol 35 ◽  
pp. 97-106
Author(s):  
Salaheddine Bakkali Yakhlef ◽  
Imane Guenoun ◽  
Benaîssa Kerdouh ◽  
Noureddine Hamamouch ◽  
Mohamed Abourouh
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Dna Isolation ◽  
Molecular Genetic Analysis ◽  
Molecular Technique ◽  
High Quality ◽  
Fresh Tissue ◽  
Dna Yield ◽  
Tetraclinis Articulata ◽  
Rapd Pcr

 English.  Molecular genetic analysis of Arar tree [Tetraclinis articulata (Vahl) Masters] is often limited by the availability of fresh tissue and an efficient and reliable protocol for high quality genomic DNA extraction. In this study, two DNA extraction protocols were specifically developed for extracting high quality genomic DNA from Arar tree leaves: modified QIAgen DNA Kit and protocol developed by Ouenzar et al. (1998). DNA yield and purity were monitored by gel electrophoresis and by determining absorbance at UV (A260/A280 and A260/A230). Both ratios were between 1.7 and 2.0, indicating that the presence of contaminating metabolites was minimal. The DNA yield obtained ranged between 20 to 40 µg/g of plant materiel. The Ouenzar and collaborators protocol gave higher yield but was more time consuming compared to QIAgen Kit. However, both techniques gave DNA of good quality that is amenable to RAPD-PCR reactions.Additionally, restriction digestion and PCR analyses of the obtained DNA showed its compatibility with downstream applications. Randomly Amplified Polymorphic DNA profiling from the isolated DNA was optimized to produce scorable and clear amplicons. The presented protocols allow easy and high quality DNA isolation for genetic diversity studies within Arar tree.Français.  Les analyses en génétique moléculaire chez le thuya de Berberie [Tetraclinis articulata (Vahl) Masters] sont souvent limitées par la disponibilité du matériel végétal frais et le temps nécessaire pour l’extraction l’ADN ainsi que par sa qualité. Dans cette étude, deux protocoles d’extraction, à partir des feuilles du thuya, de l’ADN génomique de haute qualité, ont été développés : le Kit Qiagen et le protocole mis au point par Ouenzar et al. (1998) modifiés. La qualité et la quantité de l’ADN sont évaluées par électrophorèse sur gel d’agarose et par la mesure de l’absorbance en UV à (A260/A280) et (A260/A230). Ces deux rapports varient entre 1,7 et 2,0 indiquant la faible fréquence des métabolites contaminants. Le rendement d’ADN varie entre 20 et 40 µg/g du matériel végétal. Le protocole de Ouenzar et collaborateurs donne le meilleur rendement d’ADN mais nécessite plus de temps. Néanmoins, les deux protocoles donnent un ADN de bonne qualité utilisable dans les réactions RAPD-PCR. En outre, la restriction enzymatique et l’analyse PCR de l’ADN obtenu ont montré sa compatibilité avec les applications moléculaires ultérieures. Les paramètres intervenant dans les réactions RAPD ont été optimisés. Les protocoles présentés permettent l’extraction facile de l’ADN de haute qualité nécessaire pour des études de la diversité génétique au sein du thuya.

scholarly journals A DNA extraction protocol for improved DNA yield from individual mosquitoes

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1314 ◽  
Author(s):  
Catelyn C. Nieman ◽  
Youki Yamasaki ◽  
Travis C. Collier ◽  
Yoosook Lee
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Single Individual ◽  
Dna Amount ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Genome Representation ◽  
Generation Sequencing

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

scholarly journals A cross-sectional investigation of SARS-CoV-2 seroprevalence and associated risk factors in children and adolescents in the United States

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0259823
Author(s):  
Rebecca E. Levorson ◽  
Erica Christian ◽  
Brett Hunter ◽  
Jasdeep Sayal ◽  
Jiayang Sun ◽  
...  
Keyword(s):  
Risk Factors ◽  
Children And Adolescents ◽  
Disease Transmission ◽  
Specific Antigen ◽  
Significant Risk ◽  
The United States ◽  
Mitigation Strategies ◽  
Metropolitan Region ◽  
Antibody Testing ◽  
Cross Sectional

Background Pediatric SARS-CoV-2 data remain limited and seropositivity rates in children were reported as <1% early in the pandemic. Seroepidemiologic evaluation of SARS-CoV-2 in children in a major metropolitan region of the US was performed. Methods Children and adolescents ≤19 years were enrolled in a cross-sectional, observational study of SARS-CoV-2 seroprevalence from July-October 2020 in Northern Virginia, US. Demographic, health, and COVID-19 exposure information was collected, and blood analyzed for SARS-CoV-2 spike protein total antibody. Risk factors associated with SARS-CoV-2 seropositivity were analyzed. Orthogonal antibody testing was performed, and samples were evaluated for responses to different antigens. Results In 1038 children, the anti-SARS-CoV-2 total antibody positivity rate was 8.5%. After multivariate logistic regression, significant risk factors included Hispanic ethnicity, public or absent insurance, a history of COVID-19 symptoms, exposure to person with COVID-19, a household member positive for SARS-CoV-2 and multi-family or apartment dwelling without a private entrance. 66% of seropositive children had no symptoms of COVID-19. Secondary analysis included orthogonal antibody testing with assays for 1) a receptor binding domain specific antigen and 2) a nucleocapsid specific antigen had concordance rates of 80.5% and 79.3% respectively. Conclusions A much higher burden of SARS-CoV-2 infection, as determined by seropositivity, was found in children than previously reported; this was also higher compared to adults in the same region at a similar time. Contrary to prior reports, we determined children shoulder a significant burden of COVID-19 infection. The role of children’s disease transmission must be considered in COVID-19 mitigation strategies including vaccination.

scholarly journals Food access among people who inject drugs in West Virginia

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Saba Rouhani ◽  
Sean T. Allen ◽  
Sara Whaley ◽  
Rebecca Hamilton White ◽  
Allison O’Rourke ◽  
...  
Keyword(s):  
Drug Use ◽  
West Virginia ◽  
Disease Transmission ◽  
Food Access ◽  
People Who Inject Drugs ◽  
The United States ◽  
Cross Sectional ◽  
Factors Associated ◽  
The Past ◽  
Adequate Food

Abstract Background The substance use epidemic in the United States continues to drive high levels of morbidity and mortality, particularly among people who inject drugs (PWID). Poor access to food often co-occurs with drug use and contributes to associated sequelae, such as risks for HIV and diabetes. The objective of this study was to examine factors associated with adequate food access among PWID in a rural Appalachian community. Methods Cross-sectional surveys were used to collect data among PWID aged 18 and older in Cabell County, West Virginia. Frequency of hunger and sociodemographic, structural and drug use characteristics were measured. Adequate food access was defined as reporting ‘never’ going to bed hungry at night in the past six months. Pearson’s χ2 and t-tests and multivariable logistic regression were used to identify factors associated with food access. Results Only 71 individuals (17%) reported never going to bed hungry at night in the past six months. Adjusted odds of having adequate food access were higher among PWID who completed high school (aOR 2.94; P = 0.010) and usually used drugs alone (aOR 1.97; P = 0.025), and lower among PWID who were female (aOR 0.51; P = 0.037), experienced homelessness (aOR 0.23, P < 0.001), were recently arrested (aOR 0.50 P = 0.047), and engaged in receptive sharing of injection equipment (aOR 0.52, P = 0.035). Conclusions We found extremely low food access in a population of PWID in Appalachia who are vulnerable to overdose and infectious disease transmission. Integrated interventions promoting food access are needed to improve the public health and wellbeing of people who inject drugs in Appalachia.

scholarly journals A Rapid and Inexpensive Method of DNA Extraction from Palmyra Palm (Borassus flabellifer)

2019 ◽  
pp. 1-5
Author(s):  
Soni Kumari ◽  
Ruby Rani ◽  
Jitesh Kumar ◽  
Ravi Ranjan Kumar ◽  
Tushar Ranjan
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Tween 20 ◽  
Inexpensive Method ◽  
Rapid Procedure ◽  
Dna Yield ◽  
Lysis Buffer ◽  
Yield And Purity ◽  
Different Levels

Aims: The study aims to highlight the simple optimisation, inexpensive and rapid procedure for DNA isolation from tough leaves (Palmyra palm) without compromising the yield and purity of DNA. Study Design: Leaf of palmyra palm (Borassus flabellifer) was used to conduct the experiment followed by laboratory analysis, DNA extraction and PCR amplification. Results and Discussion: The results showed that different buffers examined for the extraction of DNA provided significantly different levels of yield and purity. DNA isolated by lysis buffers C showed satisfactory amplifications in PCR. The fingerprint we obtained by using the DNA extracted by these buffers provided higher resolution than those using buffers. Conclusion: This study suggests that grinding of Palmyra palm leaves with sterile sand or cover slips and inclusion of SDS, Tween 20, and NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and β mercaptoethanol, provides a DNA yield of sufficient purity, suitable for PCR amplification and subsequent use.

scholarly journals Food Security among People who Inject Drugs in West Virginia

2021 ◽  
Author(s):  
Saba Rouhani ◽  
Sean T Allen ◽  
Sarah Whaley ◽  
Rebecca H White ◽  
Allison O'Rourke ◽  
...  
Keyword(s):  
Food Security ◽  
Drug Use ◽  
West Virginia ◽  
Disease Transmission ◽  
People Who Inject Drugs ◽  
The United States ◽  
Cross Sectional ◽  
Factors Associated ◽  
The Past ◽  
Poor Access

Abstract Background. The substance use epidemic in the United States continues to drive high levels of morbidity and mortality, particularly among people who inject drugs (PWID). Poor access to food co-occurs with illicit drug use and contributes to associated sequelae, such as HIV and diabetes. The objective of this study was to examine factors associated with food security among PWID in a rural Appalachian community. Methods. Cross-sectional surveys were used to collect data in a sample of 420 PWID aged 18 and older Cabell County, West Virginia. Frequency of food access and sociodemographic, structural and drug use characteristics were measured. Food security was defined as reporting ‘never’ going to bed hungry at night in the past six months. Pearson’s χ2 and t-tests were used to identify factors associated with food security. Multivariable logistic regression was employed to estimate adjusted odds of being food secure.Results. Only 71 individuals (17%) reported never going to bed hungry night in the past six months. Adjusted odds of being food secure were higher among PWID who completed high school (aOR 2.94; P=0.010) and usually using drugs alone (aOR 1.97; P=0.025), and lower among PWID who were female (aOR 0.51; P=0.037), experienced homelessness (aOR 0.23, P<0.001), were recently arrested (aOR 0.50 P=0.049), and engaged in receptive sharing of injection equipment (aOR 0.52, P=0.035).Conclusions. We found extremely low levels of food security in a population of PWID in Appalachia who are vulnerable to overdose and infectious disease transmission. Integrated interventions that approach food provision as harm reduction are needed to address the overlapping factors that jeopardize the health and wellbeing of people who inject drugs.

Comparison of DNA Extraction and Purification Methods from Different Soils for Metagenomic Sequencing

2014 ◽  
Vol 955-959 ◽  
pp. 306-309 ◽  
Author(s):  
Lin Hui Wu ◽  
Jian Li Liu ◽  
Jing Zeng ◽  
Ji Zhao
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Metagenomic Sequencing ◽  
Soil Dna ◽  
Dna Yield ◽  
Extraction And Purification ◽  
Purification Methods ◽  
The Impact ◽  
Different Soils

There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of soil types. This has been regarded as one of the major difficulties that hamper the development of soil microbial ecology study. No commercial nucleic acids kits currently available are capable of preparing the DNAs without modifications. The cost can be very high for DNA extraction from extreme environmental soil samples, such as soils that have extreme high or low pHs. In this work, we developed and optimized soil DNA extraction and purification methods on different soils and compared the impact of three different DNA extraction protocols on DNA yield and purity. For the three different types of soil we used, direct extraction obtained the highest DNA recover rate, but required more cleanup steps. MoBio PowerSoil® DNA Isolation Kit yields less but do not require as many downstream cleaning steps. Both of the two methods obtained a more abundant microbial community than Meta-G-NomeTMDNA Isolation Kit.

Does a Graduate Course in Fluency Disorders Make a Difference?

2010 ◽  
Vol 20 (1) ◽  
pp. 10-14 ◽  
Author(s):  
Evelyn R. Klein ◽  
Barbara J. Amster
Keyword(s):  
Undergraduate Students ◽  
Graduate Programs ◽  
The United States ◽  
Cross Sectional Study ◽  
Sectional Study ◽  
Graduate Course ◽  
Cross Sectional ◽  
Fluency Disorders ◽  
Introductory Course ◽  
Communicative Disorders

Abstract A study by Yaruss and Quesal (2002), based on responses from 134 of 239 ASHA accredited graduate programs, indicated that approximately 25% of graduate programs in the United States allow students to earn their degree without having coursework in fluency disorders and 66% of programs allow students to graduate without clinical experience treating people who stutter (PWS). It is not surprising that many clinicians report discomfort in treating PWS. This cross-sectional study compares differences in beliefs about the cause of stuttering between freshman undergraduate students enrolled in an introductory course in communicative disorders and graduate students enrolled and in the final weeks of a graduate course in fluency disorders.

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