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Sample records for sybr green qpcr

  1. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Cirera, Susanna

    2007-01-01

    Background: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high...... quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. Results: In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2...

  2. Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

    Science.gov (United States)

    Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

    2018-02-01

    TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A new whole mitochondrial genome qPCR (WMG-qPCR) with SYBR Green®to identify phlebotomine sand fly blood meals.

    Science.gov (United States)

    Rodrigues, Ana Caroline Moura; Magalhães, Rafaela Damasceno; Romcy, Kalil Andrade Mubarac; Freitas, Jeferson Lucas Sousa; Melo, Ana Carolina Fonseca Lindoso; Rodon, Fernanda Cristina Macedo; Bevilaqua, Claudia Maria Leal; Melo, Luciana Magalhães

    2017-04-30

    Phlebotomine sand flies are blood-feeding insects of marked medical and veterinary significance. Investigations on the biology of these insects hold great importance for both ecological and epidemiological purposes. The present work describes a new approach for real-time PCR (qPCR) with SYBR Green ® , named WMG-qPCR, to identify phlebotomine blood meals. The novelty of the assay was to design primers based on the Whole Mitochondrial Genome (WMG) of the potential hosts (human, dog, cat, brown rat and chicken) aiming to amplify through qPCR the regions of mitochondrial DNA (mtDNA) which are less conserved among all species. Initially, the best method for mtDNA extraction to be applied in WMG-qPCR was determined. Afterwards, amplification specificities were accessed by cross-reaction assays with mtDNA samples from all animal species, besides phlebotomine DNA. Finally, the selected primers were also tested for their limit of DNA detection through standard curves constructed by serial dilution of blood DNA obtained for each target animal species. The WMG-qPCR was able to detect as low as 10pL of blood, equivalent to 26, 84, 130, and 320fg DNA of cat, human, dog and rat, respectively. The assay was also capable to amplify as low as 5pL of chicken blood (5pg DNA). In conclusion, WMG-qPCR seems to be a promising tool to identify phlebotomine blood meals, with high species-specificity and sensitivity. Furthermore, as no supplementary techniques are required, this new approach presents minimized costs and simplified technical-training requirements for execution. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

    DEFF Research Database (Denmark)

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M. Isabel

    2011-01-01

    Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR...... 1 and 10 conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods...... was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods....

  5. Investigation of a Sybr-Green-Based Method to Validate DNA Sequences for DNA Computing

    National Research Council Canada - National Science Library

    Pogozelski, Wendy; Priore, Salvatore; Bernard, Matthew; Macula, Anthony

    2005-01-01

    .... Strands were tested for their potential to cross-hybridize by measuring fluorescence over varying temperatures in the presence of SYBR Green Strands were tested in pools to show that hybridization...

  6. Flow cytometric analysis using SYBR Green I for genome size estimation in coffee.

    Science.gov (United States)

    Ronildo Clarindo, Wellington; Roberto Carvalho, Carlos

    2011-02-01

    Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G(0)/G(1) peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide. Copyright © 2009 Elsevier GmbH. All rights reserved.

  7. Identifying Haemophilus haemolyticus and Haemophilus influenzae by SYBR Green real-time PCR.

    Science.gov (United States)

    Latham, Roger; Zhang, Bowen; Tristram, Stephen

    2015-05-01

    SYBR Green real time PCR assays for protein D (hpd), fuculose kinase (fucK) and [Cu, Zn]-superoxide dismutase (sodC) were designed for use in an algorithm for the identification of Haemophilus influenzae and H. haemolyticus. When tested on 127 H. influenzae and 60 H. haemolyticus all isolates were identified correctly. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  8. Hot electron-induced electrogenerated chemiluminescence of SYBR[reg] Green I

    International Nuclear Information System (INIS)

    Laakso, Pirita; Anttila, Hannamari; Kairisto, Veli; Eskola, Jarkko; Kulmala, Sakari; Ala-Kleme, Timo

    2005-01-01

    Hot electrons can be injected from conductor/insulator/electrolyte (C/I/E) junctions into an aqueous electrolyte solution. Injected hot electrons induce electrogenerated chemiluminescence (ECL) of various luminophores in fully aqueous solutions. Such ECL gives the basis of electrochemiluminoimmunoassays and DNA-probe assays where different luminophores can be used as electrochemiluminescent labels. This work shows that SYBR[reg] Green I is suitable as an ECL label in detection methods based on C/I/E tunnel-emission electrodes such as oxide-coated aluminium, magnesium and silicon electrodes

  9. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    Science.gov (United States)

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp.

    Science.gov (United States)

    Encinas-García, Trinidad; Mendoza-Cano, Fernando; Enríquez-Espinoza, Tania; Luken-Vega, Leonardo; Vichido-Chávez, Rodrigo; Sánchez-Paz, Arturo

    2015-02-01

    The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction.

    Science.gov (United States)

    Szuhai, K; Sandhaus, E; Kolkman-Uljee, S M; Lemaître, M; Truffert, J C; Dirks, R W; Tanke, H J; Fleuren, G J; Schuuring, E; Raap, A K

    2001-11-01

    Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogeneous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology.

  12. SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

    Science.gov (United States)

    Ghasemian, Mehrdad; Gharavi, Mohammad Javad; Akhlaghi, Lame; Mohebali, Mehdi; Meamar, Ahmad Reza; Aryan, Ehsan; Oormazdi, Hormozd; Ghayour, Zahra

    2016-03-01

    Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.

  13. Detection and characterization of Leishmania (Leishmania and Leishmania (Viannia by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

    Directory of Open Access Journals (Sweden)

    Marcello Ceccarelli

    Full Text Available Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1, whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2. The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania and Leishmania (Viannia using the qPCR2 assay followed by melting or High Resolution Melt (HRM analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania and Leishmania (Viannia subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L. infantum WHO international reference strain (MHOM/TN/80/IPT1, highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical

  14. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  15. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis.

    Science.gov (United States)

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; Garcia, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; Hermoso de Mendoza, Javier

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

  16. Development of a real-time SYBR Green PCR assay for the rapid detection of Dermatophilus congolensis

    OpenAIRE

    García, Alfredo; Martínez, Remigio; Benitez-Medina, José Manuel; Risco, David; García, Waldo Luis; Rey, Joaquín; Alonso, Juan Manuel; de Mendoza, Javier Hermoso

    2013-01-01

    Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was ...

  17. Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

    International Nuclear Information System (INIS)

    Baldock, Daniel; Nocker, Andreas; Nebe-von-Caron, Gerhard; Bongaerts, Roy

    2013-01-01

    Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR ® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR ® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pK a value. (paper)

  18. A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products.

    Science.gov (United States)

    Soares, Sónia; Amaral, Joana S; Oliveira, M Beatriz P P; Mafra, Isabel

    2013-05-01

    Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

    Science.gov (United States)

    Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

    2013-02-01

    Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Detection of Campylobacter jejuni in dairy farm environmental samples using SYBR Green real-time polymerase chain reaction.

    Science.gov (United States)

    Nam, H M; Srinivasan, V; Murinda, S E; Oliver, S P

    2005-01-01

    The aim of this study was to evaluate a SYBR Green based real-time PCR assay using well-characterized primers to detect Campylobacter jejuni in naturally contaminated dairy farm environmental samples. Specificity of the assay was determined with 62 C. jejuni strains and 120 non-C. jejuni strains. Peak melting temperature obtained with melting curves specific for C. jejuni was 77.5 degrees C. Standard curves were constructed using mean threshold cycle (C(T)) and various concentrations of C. jejuni ranging from 10(0) to 10(8) colony forming units (CFU)/mL, which resulted in a linear relationship between C(T) and log input DNA. Correlation coefficients of standard curves based on pure culture of C. jejuni in broth and spiked cells in lagoon water were R(2) = 0.995 (slope = 3.21) and R(2) = 0.988 (slope = 3.22), respectively, and sensitivity limits were 10(3) CFU/mL, respectively. After 24-h enrichment, total C. jejuni counts of all samples spiked with 10(0) CFU/mL reached >10(5) CFU/mL, and the detection limit was improved from >10(3) CFU/mL to PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of these samples being culture positive for C. jejuni. All samples that were positive by standard culture methods were also positive by the real-time PCR method. Mean C( T ) values of 48-h enriched cultures for 17 PCR-positive/culture-positive samples and eight PCR-positive/culture-negative samples were 21.4 +/- 3.6, and 34.6 +/- 1.5 (p 38.0. These results indicate that the SYBR Green real-time PCR assay provides a specific, reproducible, and simple method for detecting C. jejuni in dairy farm environmental samples.

  1. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    OpenAIRE

    Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

    2011-01-01

    Background : Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conven...

  2. The development of a rapid SYBR Green I-based quantitative PCR for detection of Duck circovirus.

    Science.gov (United States)

    Wan, Chunhe; Huang, Yu; Cheng, Longfei; Fu, Guanghua; Shi, Shao-hua; Chen, Hongmei; Peng, Chunxiang; Lin, Fang; Lin, Jiansheng

    2011-10-07

    This report describes a one-step real-time polymerase chain reaction assay based on SYBR Green I for detection of a broad range of duck circovirus (DuCV). Align with all DuCV complete genome sequences and other Genus Circovirus download from the GenBank (such as goose circovirus, pigeon circovirus), the primers targets to the replicate gene of DuCV were designed. The detection assay was linear in the range of 1.31 × 102-1.31 × 107 copies/μL. The reaction efficiency of the assay using the slope (the slope was -3.349) and the Y-intercept was 37.01 from the linear equation was estimated to be 0.99 and the correlation coefficient (R2) was 0.993. A series of experiments were carried out to assess the reproducibility, sensitivity, and specificity of the assay, following by the low intra-assay and inter-assay CVs for CT values obtained with the standard plasmids. The intra-assay CVs were equal or less than 1.89% and the inter-assay CVs were equal or less than 1.26%. There was no cross-reaction occurred with nucleic acids extracted from RA (Riemerella anatipestifer), E. coli (Escherichia coli), Duck Cholera (Pasteurella multocida), Avian influenza virus, avian paramyxovirus, Muscovy duck parvovirus, Duck reovirus, Duck hepatitis A virus as control templates. The nucleic acids extracted from samples of healthy ducks were used as negative controls. The assay was specific and reproducible. The established real time PCR was used to detect 45 DuCV-negative samples, which were tested using conventional PCR under the developed optimal conditions, each 15 for embryonated eggs, non-embryonated budgerigar eggs, newly hatched duck, the mixture of the lung, liver, spleen which were analysis for the presence of DuCV DNA, to conform that whether the DuCV can be transmitted vertically. Meanwhile, no positive result was shown by the real-time PCR method. The SYBR Green I-based quantitative PCR can therefore be practically used as an alternative diagnostic tool and a screening method

  3. APLIKASI KUANTIFIKASI KOI HERPESVIRUS : REAL TIME – QUANTITATIVE POLYMERASE CHAIN REACTION (RT-Q PCR MENGGUNAKAN SYBR GREEN PADA IKAN MAS (Cyprinus carpio

    Directory of Open Access Journals (Sweden)

    Isti Koesharyani

    2017-08-01

    , so that the existence KHV can be known exactly with high sensitivity level in fish farming.The objective of this study was to develop and evaluate the infection by Real Time Quantitative Polymerase Chain Reaction (RT- qPCR/qPCR. Sample were taken from Carp culture in West Java, West Sumatra, and South Kalimantan. The assay was done by SYBR Green RT-qPCR. The analysis result of KHV in carp revealed that the carp from Cirata-3 had the highest viral load with Ct. value 18.24 equal with 3.4 x 107 copies, and the lowest one was the carp from Banjarmasin-3 at Ct. value 33.39 (1.8 x 102 copies, while two standards plasmid and Non Template Control (NTC had Ct value of 27.24 (2 x 104copies,30.24 (2 x 103copies, and 35.65 (3.1 x 10 copies, respectively. Application KHV test by q-PCR has more advantages and sensitive than that of conventional PCR, and it can be used to detect and calculate the copy number of DNA.

  4. SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

    OpenAIRE

    Kiltie, A E; Ryan, A J

    1997-01-01

    Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This meth...

  5. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

    Directory of Open Access Journals (Sweden)

    K P Dinoop

    2016-01-01

    Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

  6. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Thiago dos Santos Gomes

    2014-01-01

    Full Text Available Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  7. Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

    Science.gov (United States)

    Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

    2003-12-15

    Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

  8. Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

    Science.gov (United States)

    Gomes, Thiago Dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Werneck de Macedo, Heloisa; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T(m)) was 73 °C and 70 °C, respectively. For E. hartmanni, the T(m) was 73 °C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

  9. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    Science.gov (United States)

    Gomes, Thiago dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T m) was 73°C and 70°C, respectively. For E. hartmanni, the T m was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  10. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    Science.gov (United States)

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6 Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6

    Directory of Open Access Journals (Sweden)

    Cynthia Liliane Motta do Canto

    2008-02-01

    Full Text Available HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL to 10-9 (equivalent to 2.46 molecules/µL. Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3' and by conventional nested PCR using primers HHV1 (outer: 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer: 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner and HHV4 (inner 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL até 10-9 (equivalente a 2,46 moleculas/µL. As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com

  12. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

    Science.gov (United States)

    Kiltie, A E; Ryan, A J

    1997-07-15

    Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.

  14. An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

    Science.gov (United States)

    Elkins, Kelly M.; Kadunc, Raelynn E.

    2012-01-01

    In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

  15. A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples

    Directory of Open Access Journals (Sweden)

    Jiyun Li

    2017-10-01

    Full Text Available The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 102 cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.

  16. Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

    Energy Technology Data Exchange (ETDEWEB)

    Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

    2003-08-01

    Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

  17. A Highly Sensitive and Selective Competition Assay for the Detection of Cysteine Using Mercury-Specific DNA, Hg2+ and Sybr Green I

    Directory of Open Access Journals (Sweden)

    Gang Liu

    2011-10-01

    Full Text Available We here report a rapid, sensitive, selective and label-free fluorescence detection method for cysteine (Cys. The conformation of mercury-specific DNA (MSD changes from a random coil form to a hairpin structure in the presence of Hg2+ due to the formation of a thymine-Hg2+-thymine (T-Hg2+-T complex. Cys can selectively coordinate with Hg2+ and extract it from the thymine-Hg2+-thymine complex. The hairpin structure dehybridizes and the fluorescence intensity of Sybr Green I (SG decreases upon addition of Cys because SG efficiently discriminates mercury-specific DNA and mercury-specific DNA/Hg2+ complex. The detection can be finished within 5 min with high sensitivity and selectivity. In addition, we can obtain variable dynamic ranges for Cys by changing the concentration of MSD/Hg2+.

  18. Modulation of the Singlet Oxygen Generation from the Double Strand DNA-SYBR Green I Complex Mediated by T-Melamine-T Mismatch for Visual Detection of Melamine.

    Science.gov (United States)

    Hu, Hao; Zhang, Jinyi; Ding, Yu; Zhang, Xinfeng; Xu, Kailai; Hou, Xiandeng; Wu, Peng

    2017-05-02

    Singlet oxygen ( 1 O 2 ), generated via photosensitization, has been proved to oxidize chromogenic substrates with neither H 2 O 2 oxidation nor enzyme (horseradish peroxidase, HRP) catalysis. Of the various methods for modulation of the 1 O 2 generation, DNA-controlled photosensitization received great attention. Therefore, integration of the formation/deformation DNA structures with DNA-controlled photosensitization will be extremely appealing in visual biosensor developments. Here, the stable melamine-thymine complex was explored in combination with DNA-controlled photosensitization for visual detection of melamine. A T-rich single stand DNA was utilized as the recognition unit. Upon the formation of the T-M-T complex, double stand DNA was formed, which was ready for the binding of SYBR Green I and activated the photosensitization. Subsequent oxidation of TMB allowed visual detection of melamine in dairy products, with spike-recoveries ranging from 94% to 106%.

  19. Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; Denayer, Sarah; Botteldoorn, Nadine; Dierick, Katelijne; De Keersmaecker, Sigrid C J; Roosens, Nancy H

    2018-04-01

    A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

  20. Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene.

    Science.gov (United States)

    Cho, Min Seok; Park, Duck Hwan; Ahn, Tae-Young; Park, Dong Suk

    2015-09-01

    The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/μl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

  1. Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9 virus.

    Directory of Open Access Journals (Sweden)

    Zheng Zhu

    Full Text Available Most recently a novel avian-origin influenza A (H7N9 virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (10(6 to 10(1 copies/ µl with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

  2. Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR

    Directory of Open Access Journals (Sweden)

    Lou Sai

    2011-07-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is a worldwide pathogen in cattle and acts as a surrogate model for hepatitis C virus (HCV. One-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-PCR assay based on SYBR Green I dye has not been established for BVDV detection. This study aims to develop a quantitative one-step RT-PCR assay to detect BVDV type-1 in cell culture. Results One-step quantitative SYBR Green I RT-PCR was developed by amplifying cDNA template from viral RNA and using in vitro transcribed BVDV RNA to establish a standard curve. The assay had a detection limit as low as 100 copies/ml of BVDV RNA, a reaction efficiency of 103.2%, a correlation coefficient (R2 of 0.995, and a maximum intra-assay CV of 2.63%. It was 10-fold more sensitive than conventional RT-PCR and can quantitatively detect BVDV RNA levels from 10-fold serial dilutions of titrated viruses containing a titer from 10-1 to 10-5 TCID50, without non-specific amplification. Melting curve analysis showed no primer-dimers and non-specific products. Conclusions The one-step SYBR Green I RT-PCR is specific, sensitive and reproducible for the quantification of BVDV in cell culture. This one-step SYBR Green I RT-PCR strategy may be further optimized as a reliable assay for diagnosing and monitoring BVDV infection in animals. It may also be applied to evaluate candidate agents against HCV using BVDV cell culture model.

  3. Relative quantification and detection of different types of infectious bursal disease virus in bursa of Fabricius and cloacal swabs using real time RT-PCR SYBR green technology

    DEFF Research Database (Denmark)

    Li, Yiping; Handberg, K.J.; Kabell, Susanne

    2007-01-01

    In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection...... or F52/70 inoculation were detected as virus positive at day I post inoculation (p.i.). The D78 viral load peaked at day 4 and day 8 p.i., while the DK01 and F52/70 viral load showed relatively high levels at day 2 p.i. In cloacal swabs, viruses detectable were at day 2 p.i. for DK01 and F52/70, day 8...... of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and beta-actin to IBDVs. Based on this P-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with D78, DK01...

  4. Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2.

    Science.gov (United States)

    Balboni, Andrea; Dondi, Francesco; Prosperi, Santino; Battilani, Mara

    2015-09-15

    Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Qualitative Sybr Green real-time detection of single nucleotide polymorphisms responsible for target-site resistance in insect pests: the example of Myzus persicae and Musca domestica.

    Science.gov (United States)

    Puggioni, V; Chiesa, O; Panini, M; Mazzoni, E

    2017-02-01

    Chemical insecticides have been widely used to control insect pests, leading to the selection of resistant populations. To date, several single nucleotide polymorphisms (SNPs) have already been associated with insecticide resistance, causing reduced sensitivity to many classes of products. Monitoring and detection of target-site resistance is currently one of the most important factors for insect pest management strategies. Several methods are available for this purpose: automated and high-throughput techniques (i.e. TaqMan or pyrosequencing) are very costly; cheaper alternatives (i.e. RFLP or PASA-PCRs) are time-consuming and limited by the necessity of a final visualization step. This work presents a new approach (QSGG, Qualitative Sybr Green Genotyping) which combines the specificity of PASA-PCR with the rapidity of real-time PCR analysis. The specific real-time detection of Cq values of wild-type or mutant alleles (amplified used allele-specific primers) allows the calculation of ΔCqW-M values and the consequent identification of the genotypes of unknown samples, on the basis of ranges previously defined with reference clones. The methodology is applied here to characterize mutations described in Myzus persicae and Musca domestica and we demonstrate it represents a valid, rapid and cost-effective technique that can be adopted for monitoring target-site resistance in field populations of these and other insect species.

  6. Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines.

    Science.gov (United States)

    Zhou, Hao; Chen, Shun; Qi, Yulin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-10-01

    Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P pathogens. © 2015 Poultry Science Association Inc.

  7. Quantification of hTERT Splice Variants in Melanoma by SYBR Green Real-time Polymerase Chain Reaction Indicates a Negative Regulatory Role for the β Deletion Variant

    Directory of Open Access Journals (Sweden)

    Lisa F. Lincz

    2008-10-01

    Full Text Available Telomerase activity is primarily determined by transcriptional regulation of the catalytic subunit, human telomerase reverse transcriptase (hTERT. Several mRNA splice variants for hTERT have been identified, but it is not clear if telomerase activity is determined by the absolute or relative levels of full-length (functional and variant hTERT transcripts. We have developed an SYBR green-based reverse transcription-quantitative polymerase chain reaction assay for the enumeration of the four common hTERT mRNA variants and correlated these with telomerase activity and telomere length in 24 human melanoma cell lines. All except five of the lines expressed four hTERT transcripts, with an overall significant level of co-occurrence between absolute mRNA levels of full-length α+/β+ hTERT and the three splice variants α-/β+, α+/β-, and α-/β-. On average, α+/β+ made up the majority (48.1% of transcripts, followed by α+/β- (44.6%, α-/β- (4.4%, and α-/β+ (2.9%. Telomerase activity ranged from 1 to 247 relative telomerase activity and correlated most strongly with the absolute amount of α+/β+ (R = 0.791, P = .000004 and the relative amount of α+/β- (R = -0.465, P = .022. This study shows that telomerase activity in melanoma cells is best determined by the absolute expression of full-length hTERT mRNA and indicates a role for the hTERT β deletion variant in the negative regulation of enzyme activity.

  8. SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

    Directory of Open Access Journals (Sweden)

    Weiping Xu

    Full Text Available A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100% and a specificity of 99.9% (2910/2912; 95%CI 99.7-100% when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

  9. SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene--a highly sensitive molecular tool for malaria parasite detection and species determination.

    Science.gov (United States)

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5-10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as 'final positive' if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5-100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7-100%) when compared against 'final positive' samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings.

  10. SYBR Green Real-Time PCR-RFLP Assay Targeting the Plasmodium Cytochrome B Gene – A Highly Sensitive Molecular Tool for Malaria Parasite Detection and Species Determination

    Science.gov (United States)

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I.; Shakely, Delér; Petzold, Max; Björkman, Anders; Mårtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/μl (p/μl) for P. falciparum and P. ovale, and 2 p/μl for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.5–10 p/μl. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as ‘final positive’ if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5–100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7–100%) when compared against ‘final positive’ samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings. PMID:25774805

  11. Optimization of rapid Salmonella enterica detection in liquid whole eggs by SYBR green I-based real-time reverse transcriptase-polymerase chain reaction.

    Science.gov (United States)

    Techathuvanan, Chayapa; D'Souza, Doris Helen

    2011-04-01

    Eggs and egg products have a high risk of Salmonella enterica serovar Enteritidis contamination leading to gastroenteritis outbreaks in humans. Thus, a rapid screening tool for viable Salmonella Enteritidis cells in the egg industry is needed. Our objective was to rapidly and sensitively detect viable Salmonella Enteritidis from spiked liquid whole eggs (LWEs) within 24 h using SYBR green I-based real-time reverse transcriptase-polymerase chain reaction (PCR) targeting the Salmonella specific invA gene along with an internal amplification control in a Bio-Rad iCycler. LWE was inoculated with Salmonella Enteritidis and mixed with tetrathionate broth, and 100 μL of serially diluted portions in phosphate-buffered saline was plated on Xylose Lysine Tergitol 4 agar or 5 mL were used for RNA extraction by the TRIzol method immediately or after enrichment of 6, 12, or 16 h at 37 °C. The real-time reverse transcriptase-PCR assay was carried out using previously described Salmonella invA gene primers. Melt temperature analysis of the PCR product was included to determine specific invA amplification. Without enrichment, the assay detection limit was 10(7) colony forming units (CFU)/25 mL LWE. After enrichment for 6 and 12 h, Salmonella Enteritidis could be detected from LWE up to 10(4) and 10(2) CFU/25 mL, respectively. Improved Salmonella Enteritidis detection up to 10(0) CFU/25 mL was obtained after 16-h enrichment. Even with 16-h enrichment, the results could be still be obtained within 24 h, which is much faster than by traditional cultural detection that takes several days. Therefore, this assay appears suitable for routine detection of Salmonella enterica contamination by the egg industry to help prevent the transmission of egg-associated Salmonella outbreaks and timely recall of contaminated products. © Mary Ann Liebert, Inc.

  12. Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil.

    Science.gov (United States)

    Romeiro, Marilia Farignoli; Souza, William Marciel de; Tolardo, Aline Lavado; Vieira, Luiz Carlos; Colombo, Tatiana Elias; Aquino, Victor Hugo; Nogueira, Maurício Lacerda; Figueiredo, Luiz Tadeu Moraes

    2016-01-01

    The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

  13. Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

    Directory of Open Access Journals (Sweden)

    Addimas Tajebe

    2015-03-01

    Conclusions: The study showed high prevalence level and fixation of Pfcrt, 76T mutation after chloroquine withdrawal. The prevalence of Pfmdr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers. However, the prevalence level was not statistically significant.

  14. A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

    Science.gov (United States)

    Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

    2010-09-01

    Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

  15. MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

    Directory of Open Access Journals (Sweden)

    Rodrigo Staggemeier

    2014-04-01

    Full Text Available A novel SYBR® green-real time polymerase chain reaction (qPCR was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  16. Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

    2017-06-01

    Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease, resulting in substantial economic losses to the swine industry worldwide. In this study, three assays, namely a conventional reverse transcription-polymerase chain reaction (RT-PCR), a SYBR Green I real-time RT-PCR and a TaqMan real-time RT-PCR targeting the highly conserved M gene of PEDV, were developed and evaluated. Then, the analytical specificity, sensitivity and reproducibility of these assays were determined and compared. The TaqMan real-time RT-PCR was 100-fold and 10,000-fold more sensitive than that of the SYBR Green I real-time RT-PCR and the conventional RT-PCR, respectively. The analytical sensitivity of TaqMan real-time RT-PCR was 10 copies/μl of target gene and no cross amplification with other viruses tested was observed. With the features of high specificity, sensitivity, and reproducibility, the TaqMan real-time RT-PCR established in this study could be a useful tool for clinical diagnosis, epidemiological surveys and outbreak investigations of PED. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Development and Characterization of a Highly Specific and Sensitive SYBR Green Reverse Transcriptase PCR Assay for Detection of the 2009 Pandemic H1N1 Influenza Virus on the Basis of Sequence Signatures▿ †

    Science.gov (United States)

    Medina, Rafael A.; Rojas, Mark; Tuin, Astrid; Huff, Stephen; Ferres, Marcela; Martinez-Valdebenito, Constanza; Godoy, Paula; García-Sastre, Adolfo; Fofanov, Yuriy; SantaLucia, John

    2011-01-01

    The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic. PMID:21084522

  18. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia.

    Science.gov (United States)

    Chaorattanakawee, Suwanna; Tyner, Stuart D; Lon, Chanthap; Yingyuen, Kritsanai; Ruttvisutinunt, Wiriya; Sundrakes, Siratchana; Sai-gnam, Piyaporn; Johnson, Jacob D; Walsh, Douglas S; Saunders, David L; Lanteri, Charlotte A

    2013-07-12

    Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of "immediate ex vivo" (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson's correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the MSF assay, when evaluating ex

  19. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

    Science.gov (United States)

    A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

  20. SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

    Science.gov (United States)

    Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

    2012-01-01

    Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

  1. Detection of allergen coding sequences of kiwi, peach, and apple in processed food by qPCR.

    Science.gov (United States)

    Graziano, Sara; Gullì, Mariolina; Marmiroli, Nelson

    2017-12-06

    Food traceability becomes lifesaving for persons suffering severe allergy or intolerance, and therefore need a complete avoidance of the immune-trigger food. This paper describes how to fingerprint the presence of some allergenic species (kiwi, peach, and apple) in foods by quantitative real-time PCR (qPCR). Five DNA extraction procedures were tested on fruits and foods. The results were statistically evaluated, and discussed. Analysis by qPCR with SYBR Green was developed to detect traces of these allergenic species in foods. Plasmids containing the target sequences of kiwi, peach and apple were employed as internal reference standard. Analysis of spiked food samples showed a limit of detection of 25 mg kg -1 for kiwi, 20 mg kg -1 for peach and 50 mg kg -1 for apple. The qPCR method here developed, combined with the use of internal plasmid reference standard, represents a specific system for the quick detection of allergenic species in complex food matrices, with a limit of detection comparable with those reported using more time-consuming methods. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Wet-lab tested microRNA assays for qPCR studies with SYBR®Green and DNA primers in pig tissues

    DEFF Research Database (Denmark)

    Mentzel, Caroline M. Junker; Skovgaard, Kerstin; Córdoba, Sarai

    2014-01-01

    MicroRNAs are key post-transcriptional regulators of gene expression that are involved in several biological processes including those that mediate disease pathophysiology. Hence, quantifying microRNA expression levels can provide important and novel insights into disease biology. In recent years...... have previously developed two useful tools in the field of microRNA quantitative real time PCR (qPCR): 1) a very specific, sensitive and simple qPCR method based on DNA primers, MiR-specific qPCR; and 2) the free primer-design software miRprimer. The present study integrates in a publicly accessible...... database all available information on validated porcine microRNA qPCR assays that have utilized these tools. Due to the high phylogenetic conservation in microRNA sequence between pig, humans and other domestic species this database is a very valuable resource for the broader scientist community who...

  3. Validation of kinetics similarity in qPCR

    Czech Academy of Sciences Publication Activity Database

    Bar, T.; Kubista, Mikael; Tichopád, Aleš

    2012-01-01

    Roč. 40, č. 4 (2012), s. 1395-1406 ISSN 0305-1048 R&D Projects: GA ČR GAP303/10/1338; GA ČR GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : REAL-TIME PCR * POLYMERASE-CHAIN- REACTION * SYBR-GREEN-I Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  4. A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

    Science.gov (United States)

    Rutledge, Robert G

    2011-03-02

    Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

  5. A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    Full Text Available BACKGROUND: Linear regression of efficiency (LRE introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. FINDINGS: Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. CONCLUSIONS: The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

  6. Detection and quantification of Spongospora subterranea f. sp. subterranea in bait plants and potato fields in Colombia using QPCR

    International Nuclear Information System (INIS)

    Garcia Bastidas, Nevar; Morales, Juan Gonzalo; Gonzalez Jaimes, Paola; Gutierrez, Pablo Andres; Marin Montoya, Mauricio

    2013-01-01

    In recent years, potato crops (Solanum tuberosum, S. phureja) have been seriously affected by powdery scab; a disease caused by Spongospora subterranea f.sp. subterranea (Sss). In Colombia, asymptomatic detection of Sss has been achieved with bait plants, PCR of its regions and ELISA tests. Unfortunately, these techniques have low sensitivity and may require long processing times. In this work, quantitative real time PCR (qPCR) was tested for detection of Sss using different sets of primers. Primers SsTQF1-SsTQR1, Spon421F-Spon494R and SscolF-SscolR (designed in this study), were tested using SYBR green, while primers sponfsponr were tested using the Taqman probe sponp. Primers Spon421F-Spon494R was discarded due to lack of specificity. Standard curves were obtained from serial dilutions of Cystosori. the 20 N. benthamiana and potato bait plants evaluated tested positive for Sss using primers SsTQF1-SsTQR1 (Ct: 10.57-29.34) and Sscolf-SscolR (Ct: 14.39-34.08) and 19 samples were positive with primers SponF-SponR-SponP, with Ct values ranging between 15,63 and 38,93. Sss was detected in 17 out of 20 root samples from potato crops in la Union (Antioquia) using primers SscolF-SscolRt with an estimated concentration of 6470 to 1,39x10 1 0 cystosori/ mL. these results suggest high levels of sss in the potato fields from this region and recall the importance of strengthening seed-certification programs in Colombia.

  7. A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

    Science.gov (United States)

    Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

    2017-10-30

    Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

  8. DETECCIÓN Y CUANTIFICACIÓN DE Spongospora subterranea f. sp. subterranea EN PLANTAS SEÑUELO Y CULTIVOS DE PAPA EN COLOMBIA MEDIANTE qPCR

    Directory of Open Access Journals (Sweden)

    Nevar Alirio García Bastidas

    2013-01-01

    Full Text Available La sarna polvosa de la papa (Solanum tuberosum , S. phureja causada por Spongospora subterranea f. sp. subterranea (Sss, es una de las enfermedades más limitantes de este cultivo. En Colombia, se han empleado diferentes métodos de detección asintomática de Sss, incluyendo bioensayos con plantas señuelo, PCR de ITS y pruebas de ELISA. Sin embargo, sus niveles de sensibilidad son bajos o requieren tiempos extensos. Una alternativa para complementar dichas herramientas es la PCR cuantitativa en tiempo real (qPCR. En este trabajo se evaluó dicha técnica utilizando los juegos de cebadores SsTQF1-SsTQR1; Spon421F-Spon494R y SscolF-SscolR (diseñados en este estudio, bajo la metodología de SYBR Green®; mientras que con Taqman® se evaluaron los cebadores SponF-SponR y la sonda SponP. Una vez determinada la funcionalidad de los cebadores, se descartó por inespecificidad, el par Spon421F-Spon494R; para los restantes se realizaron curvas estándar basadas en diluciones seriadas de quistosoros. Las pruebas de qPCR detectaron a Sss en las 20 muestras evaluadas de plantas señuelo de Nicotiana benthamiana y papa, utilizando los cebadores SsTQF1-SsTQR1 (Ct: 10,57- 29,34 y SscolF-SscolR (Ct: 14,39-34,08; mientras que 19 de las muestras fueron positivas con SponF-SponR-SponP (Ct: 15,63-38,93. A partir de 20 muestras de raíces de papa de cultivos de La Unión (Antioquia, Colombia, fue posible detectar el patógeno en 17 de ellas con SscolF-SscolR, estimándose una concentración de 6470 a 1,39 x 1010 quistorosos/mL. Estos resultados indican la ocurrencia de altos niveles de inóculo de Sss en esta región y enfatizan en la necesidad de fortalecer los programas de certificación de tubérculo-semilla en Colombia.

  9. green

    Directory of Open Access Journals (Sweden)

    Elena Grigoryeva

    2011-02-01

    Full Text Available The “green” topic follows the “youngsters”, which is quite natural for the Russian language.Traditionally these words put together sound slightly derogatory. However, “green” also means fresh, new and healthy.For Russia, and for Siberia in particular, “green” architecture does sound new and fresh. Forced by the anxious reality, we are addressing this topic intentionally. The ecological crisis, growing energy prices, water, air and food deficits… Alexander Rappaport, our regular author, writes: “ It has been tolerable until a certain time, but under transition to the global civilization, as the nature is destroyed, and swellings of megapolises expand incredibly fast, the size and the significance of all these problems may grow a hundredfold”.However, for this very severe Siberian reality the newness of “green” architecture may turn out to be well-forgotten old. A traditional Siberian house used to be built on principles of saving and environmental friendliness– one could not survive in Siberia otherwise.Probably, in our turbulent times, it is high time to fasten “green belts”. But we should keep from enthusiastic sticking of popular green labels or repainting of signboards into green color. We should avoid being drowned in paper formalities under “green” slogans. And we should prevent the Earth from turning into the planet “Kin-dza-dza”.

  10. SYBR safeTMefficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

    Science.gov (United States)

    Canela, H M S; Takami, L A; Ferreira, M E S

    2017-02-08

    Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safe TM , a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safe TM . For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safe TM . In conclusion, SYBR safe TM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

  11. Real-time PCR (qPCR) primer design using free online software.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  12. Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

    Science.gov (United States)

    Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

    2006-01-01

    Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

  13. How to Combine ChIP with qPCR.

    Science.gov (United States)

    Asp, Patrik

    2018-01-01

    Chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (qPCR) has in the last 15 years become a basic mainstream tool in genomic research. Numerous commercially available ChIP kits, qPCR kits, and real-time PCR systems allow for quick and easy analysis of virtually anything chromatin-related as long as there is an available antibody. However, the highly accurate quantitative dimension added by using qPCR to analyze ChIP samples significantly raises the bar in terms of experimental accuracy, appropriate controls, data analysis, and data presentation. This chapter will address these potential pitfalls by providing protocols and procedures that address the difficulties inherent in ChIP-qPCR assays.

  14. On non-detects in qPCR data.

    Science.gov (United States)

    McCall, Matthew N; McMurray, Helene R; Land, Hartmut; Almudevar, Anthony

    2014-08-15

    Quantitative real-time PCR (qPCR) is one of the most widely used methods to measure gene expression. Despite extensive research in qPCR laboratory protocols, normalization and statistical analysis, little attention has been given to qPCR non-detects-those reactions failing to produce a minimum amount of signal. We show that the common methods of handling qPCR non-detects lead to biased inference. Furthermore, we show that non-detects do not represent data missing completely at random and likely represent missing data occurring not at random. We propose a model of the missing data mechanism and develop a method to directly model non-detects as missing data. Finally, we show that our approach results in a sizeable reduction in bias when estimating both absolute and differential gene expression. The proposed algorithm is implemented in the R package, nondetects. This package also contains the raw data for the three example datasets used in this manuscript. The package is freely available at http://mnmccall.com/software and as part of the Bioconductor project. © The Author 2014. Published by Oxford University Press.

  15. Visualization of DNA-containing structurs in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR green I dye

    Czech Academy of Sciences Publication Activity Database

    Vítová, Milada; Hendrychová, Jana; Cepák, Vladislav; Zachleder, Vilém

    2005-01-01

    Roč. 50, č. 4 (2005), s. 333-340 ISSN 0015-5632 R&D Projects: GA ČR GA204/02/1438; GA ČR GA204/03/1113; GA AV ČR KJB5020305 Institutional research plan: CEZ:AV0Z5020903; CEZ:AV0Z60050516 Keywords : dna-containing * chlorophyta * rhodophyta Subject RIV: EE - Microbiology, Virology Impact factor: 0.918, year: 2005

  16. Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

    DEFF Research Database (Denmark)

    Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl

    2011-01-01

    . felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation...... or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp...... with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays....

  17. Diagnosis of Barmah Forest virus infection by a nested real-time SYBR green RT-PCR assay.

    Directory of Open Access Journals (Sweden)

    Linda Hueston

    Full Text Available Barmah Forest virus (BFV is a mosquito borne (+ ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR negative. Two culture-positive (neutralizing antibody negative samples were positive on first round PCR, while one sample (IgM and neutralizing antibody strongly positive, IgG negative was positive on second round PCR, suggesting that viral RNA is detectable and transiently present in early infection. PCR can provide results faster than culture, is capable of high throughput and by sequencing the PCR product strain variants can be characterized.

  18. Molecular detection of Phytophthora ramorum by real-time PCR using Taqman, SYBR Green and molecular beacons with three genes

    Science.gov (United States)

    G.J. Bilodeau; C.A. Lévesque; A.W.A.M. De Cock; C. Duchaine; G. Kristjansson; R.C. Hamelin

    2006-01-01

    Sudden oak death, caused by Phytophthora ramorum, is a severe disease that can affect numerous species of trees and shrubs. This pathogen has been spread via nursery stock, and quarantine measures are currently in place to prevent further spread. Molecular assays have been developed to rapidly detect and identify P. ramorum, but...

  19. Diagnosis of Barmah Forest Virus Infection by a Nested Real-Time SYBR Green RT-PCR Assay

    OpenAIRE

    Hueston, Linda; Toi, Cheryl S.; Jeoffreys, Neisha; Sorrell, Tania; Gilbert, Gwendolyn

    2013-01-01

    Barmah Forest virus (BFV) is a mosquito borne (+) ssRNA alphavirus found only in Australia. It causes rash, myalgia and arthralgia in humans and is usually diagnosed serologically. We developed a real-time PCR assay to detect BFV in an effort to improve diagnosis early in the course of infection. The limit of detection was 16 genome equivalents with a specificity of 100%. Fifty five serum samples from BFV-infected patients were tested by the PCR. 52 of 53 antibody-positive samples were PCR ne...

  20. A novel strategy for human papillomavirus detection and genotyping with SybrGreen and molecular beacon polymerase chain reaction

    NARCIS (Netherlands)

    Szuhai, K; Sandhaus, E; Kolkman-Uljee, SM; Lemaitre, M; Truffert, JC; Dirks, RW; Tanke, HJ; Fleuren, GJ; Schuuring, E; Raap, AK

    2001-01-01

    Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for

  1. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    Science.gov (United States)

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  2. Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

    Directory of Open Access Journals (Sweden)

    Dial Stacey L

    2008-07-01

    Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

  3. Removal of between-run variation in a multi-plate qPCR experiment

    NARCIS (Netherlands)

    Ruijter, Jan M.; Ruiz Villalba, Adrián; Hellemans, Jan; Untergasser, Andreas; van den Hoff, Maurice J. B.

    2015-01-01

    Quantitative PCR (qPCR) is the method of choice in gene expression analysis. However, the number of groups or treatments, target genes and technical replicates quickly exceeds the capacity of a single run on a qPCR machine and the measurements have to be spread over more than 1 plate. Such

  4. RDML-Ninja and RDMLdb for standardized exchange of qPCR data

    NARCIS (Netherlands)

    Ruijter, Jan M.; Lefever, Steve; Anckaert, Jasper; Hellemans, Jan; Pfaffl, Michael W.; Benes, Vladimir; Bustin, Stephen A.; Vandesompele, Jo; Untergasser, Andreas

    2015-01-01

    The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that

  5. Towards green loyalty: the influences of green perceived risk, green image, green trust and green satisfaction

    Science.gov (United States)

    Chrisjatmiko, K.

    2018-01-01

    The paper aims to present a comprehensive framework for the influences of green perceived risk, green image, green trust and green satisfaction to green loyalty. The paper also seeks to account explicitly for the differences in green perceived risk, green image, green trust, green satisfaction and green loyalty found among green products customers. Data were obtained from 155 green products customers. Structural equation modeling was used in order to test the proposed hypotheses. The findings show that green image, green trust and green satisfaction has positive effects to green loyalty. But green perceived risk has negative effects to green image, green trust and green satisfaction. However, green perceived risk, green image, green trust and green satisfaction also seems to be a good device to gain green products customers from competitors. The contributions of the paper are, firstly, a more complete framework of the influences of green perceived risk, green image, green trust and green satisfaction to green loyalty analyses simultaneously. Secondly, the study allows a direct comparison of the difference in green perceived risk, green image, green trust, green satisfaction and green loyalty between green products customers.

  6. Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

    OpenAIRE

    Andersen, Sandra Christine; Fachmann, Mette Sofie Rousing; Kiil, Kristoffer; Møller Nielsen, Eva; Hoorfar, Jeffrey

    2017-01-01

    In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample prepar...

  7. Green(ing) infrastructure

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2014-03-01

    Full Text Available (Krayenhoff and Bass, 2003; Foster, Lowe and Winkelman, 2012; Gill, 2007).spell out all authors first time referenced Biophilic urbanism and green infrastructure Harvard biologist E. O. Wilson popularised the concept of biophilia, describing it as “the...

  8. HF183/BFDrev and HumM2 qPCR data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Concentration estimates for HF183/BFDrev and HumM2 qPCR genetic markers in raw sewage collected from 54 geographic locations across the United States. This dataset...

  9. MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples

    Science.gov (United States)

    DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from ...

  10. Pathogens in Maternal Blood and Fetal Cord Blood Using Q-Pcr Assay

    OpenAIRE

    Guang Qiong Hou; Sidney S. Chen; Chan Ping Lee

    2006-01-01

    Objective: To evaluate the rates of infection of asymptomatic fetuses and mothers through fetal cord blood and maternal blood examination results. Materials and Methods: Quantitative PCR (Q-PCR) was used to detect pathogens in maternal peripheral blood and fetal cord blood after delivery of term pregnancy at Buddhist Tzu Chi Medical Center, Hualien, between July 2002 and June 2003. Results: We used Q-PCR to detect pathogens in 29 samples of maternal blood. The maternal hepatitis B virus...

  11. QPCR: Application for real-time PCR data management and analysis

    Directory of Open Access Journals (Sweden)

    Eichhorn Heiko

    2009-08-01

    Full Text Available Abstract Background Since its introduction quantitative real-time polymerase chain reaction (qPCR has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at http://genome.tugraz.at/QPCR

  12. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments.

    Science.gov (United States)

    Ganger, Michael T; Dietz, Geoffrey D; Ewing, Sarah J

    2017-12-01

    qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed. Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log 10 (E) ∙ C q , which we call the efficiency-weighted C q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation. The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

  13. Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

    Science.gov (United States)

    Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Shively, Dawn A.; Nevers, Meredith B.

    2010-01-01

    Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) (R = 0.650, P N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher (P R = 0.565, P N = 32). Culturable E. coli and enterococci densities were significantly correlated (R = 0.682, P N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results

  14. Metaphysical green

    OpenAIRE

    Earon, Ofri

    2011-01-01

    “Sensation of Green is about the mental process like touching, seeing, hearing, or smelling, resulting from the immediate stimulation of landscape forms, plants, trees, wind and water. Sensation of Green triggers a feeling of scale, cheerfulness, calmness and peace. The spatial performance of Sensation of Green is created by a physical interaction between the language of space and the language of nature” The notion of Sensation of Green was developed through a previous study ‘Learning from th...

  15. Green Tourism

    OpenAIRE

    Hasan, Ali

    2014-01-01

    Green tourism is defined as environmentally friendly tourism activities with various focuses and meanings. In a broad term, green tourism is about being an environmentally friendly tourist or providing environmentally friendly tourist services. The green tourism concept would be highly appealing to tourism enterprises and operators owing to increasing governmental pressure to improve environmental performance by adopting effective and tangible environmental management techniques. Green to...

  16. Legionella detection by culture and qPCR: Comparing apples and oranges.

    Science.gov (United States)

    Whiley, Harriet; Taylor, Michael

    2016-01-01

    Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

  17. Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

    Science.gov (United States)

    Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

    2016-01-01

    The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

  18. Riboflavin transporter-2 (rft-2) of Caenorhabditis elegans: Adaptive ...

    Indian Academy of Sciences (India)

    2015-04-27

    Apr 27, 2015 ... Finnzymes, USA. Maxima SYBR Green/ROX qPCR. Master Mix was purchased from Thermo Scientific, USA. Moloney Murine Leukemia Virus Reverse Transcriptase and restriction enzymes were purchased from Fermentas,. USA. DNA oligonucleotide primers were ordered from. Sigma-Aldrich (Bangalore ...

  19. Green Beans

    OpenAIRE

    Sherwood, Brianne; Inman, Elizabeth

    2011-01-01

    Green beans are thought to have originated in Peru and spread through South and Central America by Indian tribes. Spanish explorers introduced them into Europe in the 16th century. Surveys indicate that 60% of commercially grown green beans are produced in the United States. Particularly, Illinois, Michigan, New York, and Wisconsin produce the greatest amount of green beans in the United States.

  20. Identification of Listeria monocytogenes on Green Mussels (Perna viridis and Cockle Shell (Anadara granosa

    Directory of Open Access Journals (Sweden)

    Winiati Puji Rahayu

    2016-12-01

    Full Text Available Green mussel (Perna viridis and cockle shell (Anadara granosa are one of many sources of animalprotein which is many cultivated in Indonesia because their price is relatively affordable. This study wasconducted to identify the presence of Listeria monocytogenes in 27 samples of green mussels and 3 samplesof cockle shells using real-time Polymerase Chain Reaction (real-time PCR and biochemical methods. Thetarget gene for amplification in real-time PCR was an hlyA gene because this gene was a determinant ofvirulence genes that produce listeriolysin O. Primers used in this study were forward primer DG69 (GTGCCG GGT AAA AGA CCA TA and reverse primer DG74 (CGC CAC TGA GAT ACT AT and fluorescencesignals indicator using SYBR Green I. The results of analysis using real-time PCR were negative Listeriamonocytogenes in all samples, while using biochemical methods there was one of 30 samples contaminatedby Listeria welshimeri.

  1. Metaphysical green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    example is a tiny Danish summer house from 1918 . The second example is ‘House before House’ , in Tokyo. The third example is a prefabricated house ‘CHU’ . The analysis evaluates the characteristics of diverse tones of green – from green image to green sensation. The analysis is based on the original...... of Sensation of Green is created by a physical interaction between the language of space and the language of nature” The notion of Sensation of Green was developed through a previous study ‘Learning from the Summer House’ investigating the unique architectural characteristics of the Danish summer houses...

  2. Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

    Directory of Open Access Journals (Sweden)

    María-José Chapela

    2015-12-01

    Full Text Available Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article.

  3. Metaphysical green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    example is a tiny Danish summer house from 1918 . The second example is ‘House before House’ , in Tokyo. The third example is a prefabricated house ‘CHU’ . The analysis evaluates the characteristics of diverse tones of green – from green image to green sensation. The analysis is based on the original...... of Sensation of Green is created by a physical interaction between the language of space and the language of nature” The notion of Sensation of Green was developed through a previous study ‘Learning from the Summer House’ investigating the unique architectural characteristics of the Danish summer houses...... the Sensation of Green? Three existing examples are agents to this discussion. The first example is a Danish summer house. The other two are international urban examples. While the summer house articulates the original meaning of Sensation of Green, the urban examples illustrate its urban context. The first...

  4. Etiological profile of early neonatal bacterial sepsis by multiplex qPCR.

    Science.gov (United States)

    Silva-Junior, Walter P; Martins, Almir S; Xavier, Paula C N; Appel, Kelly L A; Oliveira Junior, Silvio A; Palhares, Durval B

    2016-12-30

    Given the major impact in terms of morbidity and mortality that episodes of early neonatal sepsis (ENS) have on both newborns and health systems, this study aimed to identify the etiological profile of early neonatal bacterial sepsis by a multiplex quantitative real-time polymerase chain reaction (qPCR). Blood samples from newborns diagnosed with clinical ENS and hospitalized in neonatal intensive care units (NICUs) were collected and analyzed using the multiplex qPCR method to detect Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter sp., Serratia sp., and Staphylococcus aureus. A universal primer was used in the analysis. A total of 150 neonates with clinical sepsis and 10 newborns as healthy controls were included in the study. The group with clinical sepsis was 100% positive for the presence of bacterial genomic DNA through the universal primer. The control group showed negativity by qPCR. The multiplex qPCR analysis showed that 76% of the samples were positive for Escherichia coli, 34% for Staphylococcus aureus, 13.3% for Streptococcus agalactiae, 7.3% for Pseudomonas aeruginosa, and 0.7% for Enterobacter sp. and Serratia sp. Multiplex qPCR of patients with clinical sepsis matched with 8.1% of the blood samples that tested positive by the microbiological method. Rapid and sensitive detection of the pathogens causing ENS by this new multi-target approach based on multiplex qPCR could potentially excel compared to microbiological methods, with the simple objective of facilitating the progression to a more rapid and specific antimicrobial therapy, avoiding the abuse of antibiotics in NICUs.

  5. Green Chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Collison, Melanie

    2011-05-15

    Green chemistry is the science of chemistry used in a way that will not use or create hazardous substances. Dr. Rui Resendes is working in this field at GreenCentre Canada, an offshoot of PARTEQ Innovations in Kingston, Ontario. GreenCentre's preliminary findings suggest their licensed product {sup S}witchable Solutions{sup ,} featuring 3 classes of solvents and a surfactant, may be useful in bitumen oil sands extraction.

  6. Quantification of Las gene by qPCR from orange juice extracted from Huanglongbing infected fruit

    Science.gov (United States)

    The objective of this research is to establish a methodology to quantify the Candidatus Liberibacter asiaticus (CLas) in orange juice as an indicator of orange juice quality. Current standard method for citrus Huanglongbing (HLB) diagnosis is using real-time Polymerase Chain Reaction (qPCR) to quan...

  7. Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

    Science.gov (United States)

    Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

  8. Pathogens in Maternal Blood and Fetal Cord Blood Using Q-Pcr Assay

    Directory of Open Access Journals (Sweden)

    Guang Qiong Hou

    2006-06-01

    Conclusion: Our results revealed an unexpectedly high incidence of pathogens in fetal cord blood. Screening for the above pathogens in donor cord blood in cord blood banks using Q-PCR is strongly urged to decrease morbidity and mortality rates in fetal cord blood stem cell transplant recipients.

  9. Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

    Science.gov (United States)

    Harshman, Dustin K.; Rao, Brianna M.; McLain, Jean E.; Watts, George S.; Yoon, Jeong-Yeol

    2015-01-01

    Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections. PMID:26601245

  10. Spatial expression profiles in the Xenopus laevis oocytes measured with qPCR tomography

    Czech Academy of Sciences Publication Activity Database

    Šindelka, R.; Šídová, Monika; Švec, David; Kubista, Mikael

    2010-01-01

    Roč. 51, - (2010), s. 87-91 ISSN 1046-2023 R&D Projects: GA AV ČR(CZ) IAA500970904; GA ČR GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : Xenopus * Oocyte * qPCR tomography Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  11. Detection and quantification limits of the EPA Enterococcus qPCR method

    Science.gov (United States)

    The U.S. EPA will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality in 2013 and has published preliminary proposed water quality criteria guidelines for the method. An im...

  12. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  13. The need for transparency and good practices in the qPCR literature

    DEFF Research Database (Denmark)

    Bustin, Stephen A; Benes, Vladimir; Garson, Jeremy

    2013-01-01

    Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication...

  14. QPCR quantification of ammonia oxidizing bacteria: What should the target be?

    DEFF Research Database (Denmark)

    Musovic, Sanin; Palomo, Alejandro; Diwan, Vaibhav

    be carried out by targeting either the 16S rRNA gene or amoA, for which standard primer sets are widely used. Using these two approaches to quantify AOB abundance across three Danish rapid sand filters (RSFs) revealed a significant discrepancy: in two RSFs, the amoA-based qPCR consistently yielded estimate...

  15. Green lights

    DEFF Research Database (Denmark)

    Fisker, Peter Kielberg

    variation in greenness is positively associated with year-on-year changes in luminosity: If a unit of observation experiences a predicted variation in greenness that lies 1 standard deviation below the global mean, on average 1.5 - 2.5 light pixels out of 900 are extinguished that year. Finally, an attempt...... as greenness estimated by lagged variation in monthly rainfall and temperature. This definition of drought performs well in identifying self-reported drought events since 2000 compared with measures of drought that do not take greenness into account, and the subsequent analysis indicates that predicted...

  16. Why the need for qPCR publication guidelines?--The case for MIQE.

    Science.gov (United States)

    Bustin, Stephen A

    2010-04-01

    The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the "Materials and Methods" sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments ("MIQE"). MIQE aims to restructure to-day's free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology. Copyright 2009 Elsevier Inc. All rights reserved.

  17. Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2006-12-01

    Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

  18. Identification by real-time PCR with SYBR Green of Leishmania spp. and Serratia marcescens in canine 'sterile' cutaneous nodular lesions.

    Science.gov (United States)

    Cornegliani, Luisa; Corona, Antonio; Vercelli, Antonella; Roccabianca, Paola

    2015-06-01

    Noninfectious, non-neoplastic, nodular to diffuse, so-called 'sterile' granulomatous/pyogranulomatous skin lesions (SGPSLs) are infrequently identified in dogs and may represent a diagnostic challenge. Their correct identification is based on history, histopathology and absence of intralesional foreign bodies and micro-organisms. The aim of this study was to investigate the presence of Leishmania spp., Mycobacterium spp., Serratia marcescens and Nocardia spp. by real-time PCR in canine nodular skin lesions histologically diagnosed as putatively sterile. Formalin-fixed skin biopsies were collected from 40 dogs. All samples were associated with an SGPSL diagnosis characterized by multifocal, nodular to diffuse, periadnexal and perifollicular pyogranulomas/granulomas. Neither micro-organisms nor foreign bodies were detected with haematoxylin and eosin staining, under polarized light. Further analyses included periodic acid Schiff, Ziehl-Neelsen, Fite Faraco, Giemsa and Gram histochemical stains; anti-Bacillus Calmette-Guérin (BCG) and Leishmania spp. immunohistochemistry; and real-time PCR analysis for Leishmania spp., Mycobacterium spp., S. marcescens and Nocardia spp. Special stains and BCG/immunohistochemistry were negative in all samples. Real-time PCR was positive for Leishmania spp. in four of 40 biopsies and for S. marcescens in two of 40 samples. Real-time PCR for Mycobacterium spp. and Nocardia spp. was negative. No correlation between real-time PCR positivity and a specific histological pattern was identified. Leishmania spp. have been previously identified as possible agents of certain SGPSLs, while the involvement of S. marcescens has not been investigated previously. According to our findings, Serratia spp. should be included in the list of agents possibly associated with a subgroup of granulomatous/pyogranulomatous skin lesions in dogs. © 2015 ESVD and ACVD.

  19. Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.

    Science.gov (United States)

    Gentilini, Fabio; Turba, Maria E

    2014-01-01

    A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

    Directory of Open Access Journals (Sweden)

    Boboli Hedwige

    2010-09-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR for the detection of P. aeruginosa in respiratory samples of not chronically infected CF patients. Results In this national study, we followed CF patients during periods between 1 to 15 months. For a total of 852 samples, 729 (86% remained P. aeruginosa negative by both culture and qPCR, whereas 89 samples (10% were positive by both culture and qPCR. Twenty-six samples were negative by culture but positive by qPCR, and 10 samples were positive by culture but remained negative by qPCR. Five of the 26 patients with a culture negative, qPCR positive sample became later P. aeruginosa positive both by culture and qPCR. Conclusion Based on the results of this study, it can be concluded that qPCR may have a predictive value for impending P. aeruginosa infection for only a limited number of patients.

  1. Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results?

    NARCIS (Netherlands)

    Dijkstra, J.R.; Kempen, L.C.L.T. van; Nagtegaal, I.D.; Bustin, S.A.

    2014-01-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published.

  2. Green thermodynamics

    Energy Technology Data Exchange (ETDEWEB)

    Cengel, Y.A. [Nevada Univ., Reno, NV (United States). Dept. of Mechanical Engineering

    2006-07-01

    Green components of thermodynamics were identified and general aspects of green practices associated with thermodynamics were assessed. Energy uses associated with fossil fuels were reviewed. Green energy sources such as solar, wind, geothermal and hydropower were discussed, as well as biomass plantations. Ethanol production practices were reviewed. Conservation practices in the United States were outlined. Energy efficiency and exergy analyses were discussed. Energy intensity measurements and insulation products for houses were also reviewed. Five case studies were presented to illustrate aspects of green thermodynamics: (1) light in a classroom; (2) fuel saved by low-resistance tires; and (3) savings with high-efficiency motors; (4) renewable energy; and (5) replacing a valve with a turbine at a cryogenic manufacturing facility. It was concluded that the main principles of green thermodynamics are to ensure that all material and energy inputs minimize the depletion of energy resources; prevent waste; and improve or innovate technologies that achieve sustainability. 17 refs., 2 tabs., 9 figs.

  3. Green consumerism

    DEFF Research Database (Denmark)

    de Groot, Judith I.M.; Schuitema, Geertje; Garson, Carrie Lee

    and biospheric values influence the importance of such ‘green’ product characteristics on purchasing intentions. In two within-subjects full-factorial experimental studies (N = 100 and N = 107), we found that purchase intentions of products were only steered by green characteristics if prices were low......Our presentation will focus on the influence of product characteristics and values on green consumerism. Although generally a majority of consumers support the idea of purchasing green products, we argue, based on social dilemma theory, that proself product characteristics and egoistic...... and the brand was familiar. Green product characteristics did not influence purchase intentions at all when these proself product characteristics were not fulfilled (i.e., high prices and unfamiliar brands). The importance of proself and green product characteristics on purchasing intentions was also...

  4. Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

    DEFF Research Database (Denmark)

    Andersen, Sandra Christine; Fachmann, Mette Sofie Rousing; Kiil, Kristoffer

    2017-01-01

    In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do...... not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked...... with 10³ or 10⁶ colony-forming units (CFU)/g Campylobacter jejuni, as well as porcine fecal samples spiked with 10³ or 10⁶ CFU/g Salmonella typhimurium. DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA...

  5. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

    Science.gov (United States)

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D.; Pereira, Luiz Cezar M.; Silva, Ita de Oliveira e; Ruiz-Miranda, Carlos R.; Truman, Richard; Stone, Anne C.

    2015-01-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts. PMID:26571269

  6. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    Science.gov (United States)

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D; Pereira, Luiz Cezar M; Silva, Ita de Oliveira; Ruiz-Miranda, Carlos R; Truman, Richard; Stone, Anne C

    2015-11-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

  7. Detection of Streptococcus equi subspecies equi using a triplex qPCR assay.

    Science.gov (United States)

    Webb, Katy; Barker, Colin; Harrison, Tihana; Heather, Zoe; Steward, Karen F; Robinson, Carl; Newton, J Richard; Waller, Andrew S

    2013-03-01

    Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

    OpenAIRE

    Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D.; Pereira, Luiz Cezar M.; Silva, Ita de Oliveira e; Ruiz-Miranda, Carlos R.; Truman, Richard; Stone, Anne C.

    2015-01-01

    Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were ...

  9. Green Infrastructure

    Science.gov (United States)

    To promote the benefits of green infrastructure, help communities overcome barriers to using GI, and encourage the use of GI to create sustainable and resilient water infrastructure that improves water quality and supports and revitalizes communities.

  10. Green Roofs

    Energy Technology Data Exchange (ETDEWEB)

    None

    2004-08-01

    A New Technology Demonstration Publication Green roofs can improve the energy performance of federal buildings, help manage stormwater, reduce airborne emissions, and mitigate the effects of urban heat islands.

  11. Green Tea

    Science.gov (United States)

    ... on April 16, 2015. [Database subscription]. Hartley L, Flowers N, Holmes J, et al. Green and black ... health care provider. The mention of any product, service, or therapy is not an endorsement by NCCIH. ...

  12. Green Engineering

    Science.gov (United States)

    Green Engineering is the design, commercialization and use of processes and products that are feasible and economical while reducing the generation of pollution at the source and minimizing the risk to human health and the environment.

  13. qPCR analysis of bivalve larvae feeding preferences when grazing on mixed microalgal diets.

    Directory of Open Access Journals (Sweden)

    Kai Liao

    Full Text Available Characterization of the feeding preferences of bivalve larvae would help improving the bivalve aquaculture and hatchery by providing appropriate microalgal diets. However, inaccurate and laborious identification and counting of microalgal species have challenged the selective feeding of bivalves. In the present study, we developed a highly specific and sensitive assay using quantitative polymerase chain reaction (qPCR to assess the selective feeding of bivalve larvae based on species-specific primers targeting to microalgal 18S rDNA sequences. The assay exhibited good specificity. The detection limits of the qPCR assay were 769, 71, 781 and 21 18S rDNA copies for Chaetoceros calcitrans, Isochrysis galbana, Platymonas helgolandica and Nannochloropsis oculata, respectively. Using such assay, we found that C. calcitrans and I. galbana were preferentially ingested, whereas N. oculata was preferentially rejected in biodeposits of four bivalve species, Tegillarca gransa, Cyclina sinensis, Scapharca subcrenata and Sinonovacula constricta. Furthermore, our growth experiments revealed that C. calcitrans and I. galbana could significantly promote the shell growth, whereas feeding of N. oculata resulted in poorer growth of four bivalve species. These data indicated that qPCR might be useful in screening of efficient and reliable microalgal species for each bivalve species, leading to improved bivalve aquaculture and hatchery.

  14. Quantification of Salmonella Typhi in water and sediments by molecular-beacon based qPCR.

    Science.gov (United States)

    Rani, Neetika; Vajpayee, Poornima; Bhatti, Saurabh; Singh, Smriti; Shanker, Rishi; Gupta, Kailash Chand

    2014-10-01

    A molecular-beacon based qPCR assay targeting staG gene was designed for specific detection and quantification of S. Typhi and validated against water and sediment samples collected from the river Ganga, Yamuna and their confluence on two days during Mahakumbha mela 2012-2013 (a) 18 December, 2012: before six major religious holy dips (Makar Sankranti, Paush Poornima, Mauni Amavasya, Basant Panchami, Maghi Poornima and Mahashivratri) (b) 10 February, 2013: after the holy dip was taken by over 3,00,00,000 devotees led by ascetics of Hindu sects at Sangam on 'Mauni Amavasya' (the most auspicious day of ritualistic mass bathing). The assay could detect linearly lowest 1 genomic equivalent per qPCR and is highly sensitive and selective for S. Typhi detection in presence of non specific DNA from other bacterial strains including S. Paratyphi A and S. Typhimurium. It has been observed that water and sediment samples exhibit S. Typhi. The mass holy dip by devotees significantly affected the water and sediment quality by enhancing the number of S. Typhi in the study area. The qPCR developed in the study might be helpful in planning the intervention and prevention strategies for control of enteric fever outbreaks in endemic regions. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

    Science.gov (United States)

    Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

    2014-02-15

    A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping.

    Science.gov (United States)

    Mehta, Bhavik; Venables, Samantha; Roffey, Paul

    2018-01-01

    Massively parallel sequencing (MPS) is fast approaching operational use in forensic science, with the capability to analyse hundreds of DNA identity and DNA intelligence markers in multiple samples simultaneously. The ForenSeq™ DNA Signature Kit on MiSeq FGx™ (Illumina) workflow can provide profiles for autosomal short tandem repeats (STRs), X chromosome and Y chromosome STRs, identity single nucleotide polymorphisms (SNPs), biogeographical ancestry SNPs and phenotype (eye and hair colour) SNPs from a sample. The library preparation procedure involves a series of steps including target amplification, library purification and library normalisation. This study highlights the comparison between the manufacturer recommended magnetic bead normalisation and quantitative polymerase chain reaction (qPCR) methods. Furthermore, two qPCR chemistries, KAPA® (KAPA Biosystems) and NEBNext® (New England Bio Inc.), have also been compared. The qPCR outperformed the bead normalisation method, while the NEBNext® kit obtained higher genotype concordance than KAPA®. The study also established an MPS workflow that can be utilised in any operational forensic laboratory.

  17. Evaluation of qPCR and plate counting for quantifying thermophilic starters in cheese.

    Science.gov (United States)

    Achilleos, Christine; Berthier, Françoise

    2017-08-01

    The respective inputs of plate counting and qPCR for the quantification of starters in cheese were evaluated using hard-cooked cheeses made with various starter combinations. Five starter strains were quantified at their different growth phases, from 0.5 h to day 214 of manufacture: one strain of Streptococcus thermophilus (ST) and two strains each of Lactobacillus delbrueckii (LD) and Lactobacillus helveticus (LH). Numbers of colony-forming units (CFU) were obtained by plate counting (PC) and qPCR (GN CFU ). The qPCR standard curves require a special attention since GN CFU depends on the degree of culturability of the standard culture. Discrepancies were evidenced from the vat milk to the end of ripening. During cheese making, GN CFU were lower than PC at the inoculation for all ST and LD samples and 83% of the LH samples, and during both the exponential and stationary phases for many of the ST and LD samples. During ripening which corresponds to the decline phase, GN CFU were higher than PC for 90% of the ST, 78% of the LD and 69% of the LH samples. Hypotheses are discussed to explain those discrepancies. The data provided by GN CFU and PC complement each other, providing a better description of starter growth in cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Green lasers

    DEFF Research Database (Denmark)

    Jensen, Ole Bjarlin

    2010-01-01

    Well over a dozen papers at this year's Photonics West meeting in San Francisco boasted improvements in harmonic generation to produce visible laser beams, most of them in the green spectral range......Well over a dozen papers at this year's Photonics West meeting in San Francisco boasted improvements in harmonic generation to produce visible laser beams, most of them in the green spectral range...

  19. Identification of Listeria monocytogenes on Green Mussels and Cockle Shell

    Directory of Open Access Journals (Sweden)

    Winiati Puji Rahayu

    2017-02-01

    Full Text Available AbstractGreen mussel (Perna viridis and cockle shell (Anadara granosa are one of many sources of animal protein which is many cultivated in Indonesia because their price is relatively affordable. This study was conducted to identify the presence of Listeria monocytogenes in 27 samples of green mussels and 3 samples of cockle shells using real-time Polymerase Chain Reaction (real-time PCR and biochemical methods. The target gene for amplification in real-time PCR was an hlyA gene because this gene was a determinant of virulence genes that produce listeriolysin O. Primers used in this study were forward primer DG69 (GTG CCG GGT AAA AGA CCA TA and reverse primer DG74 (CGC CAC TGA GAT ACT AT and fluorescence signals indicator using SYBR Green I. The results of analysis using real-time PCR were negative Listeria monocytogenes in all samples, while using biochemical methods there was one of 30 samples contaminated by Listeria welshimeri.

  20. Sources of variability in the measurement of Ascaris lumbricoides infection intensity by Kato-Katz and qPCR.

    Science.gov (United States)

    Easton, Alice V; Oliveira, Rita G; Walker, Martin; O'Connell, Elise M; Njenga, Sammy M; Mwandawiro, Charles S; Webster, Joanne P; Nutman, Thomas B; Anderson, Roy M

    2017-05-25

    Understanding and quantifying the sources and implications of error in the measurement of helminth egg intensity using Kato-Katz (KK) and the newly emerging "gold standard" quantitative polymerase chain reaction (qPCR) technique is necessary for the appropriate design of epidemiological studies, including impact assessments for deworming programs. Repeated measurements of Ascaris lumbricoides infection intensity were made from samples collected in western Kenya using the qPCR and KK techniques. These data were combined with data on post-treatment worm expulsions. Random effects regression models were used to quantify the variability associated with different technical and biological factors for qPCR and KK diagnosis. The relative precision of these methods was compared, as was the precision of multiple qPCR replicates. For both KK and qPCR, intensity measurements were largely determined by the identity of the stool donor. Stool donor explained 92.4% of variability in qPCR measurements and 54.5% of observed measurement variance for KK. An additional 39.1% of variance in KK measurements was attributable to having expelled adult A. lumbricoides worms following anthelmintic treatment. For qPCR, the remaining 7.6% of variability was explained by the efficiency of the DNA extraction (2.4%), plate-to-plate variability (0.2%) and other residual factors (5%). Differences in replicate measurements by qPCR were comparatively small. In addition to KK variability based on stool donor infection levels, the slide reader was highly statistically significant, although it only explained 1.4% of the total variation. In a comparison of qPCR and KK variance to mean ratios under ideal conditions, the coefficient of variation was on average 3.6 times larger for KK highlighting increased precision of qPCR. Person-to-person differences explain the majority of variability in egg intensity measurements by qPCR and KK, with very little additional variability explained by the technical factors

  1. Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

    DEFF Research Database (Denmark)

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo

    2015-01-01

    The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays...... for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined...... in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes...

  2. Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

    DEFF Research Database (Denmark)

    Krøjgaard, Louise H.; Krogfelt, Karen A.; Albrechtsen, Hans-Jorgen

    2011-01-01

    Background: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant...... temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool...... detection limit (> 10 CFU/L) to 1.6*10(6) CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively...

  3. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Amin Forootan

    2017-06-01

    Full Text Available Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD and the limit of quantification (LoQ as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

  4. Construction of Genetically Engineered Streptococcus gordonii Strains to Provide Control in QPCR Assays for Assessing Microbiological Quality in Recreational Water.

    Science.gov (United States)

    Quantitative PCR (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for...

  5. Green banking

    Directory of Open Access Journals (Sweden)

    Maja Drobnjaković

    2013-06-01

    Full Text Available There is an urgent need to march towards “low - carbon economy”. Global challenges of diminishing fossil fuel reserves, climate change, environmental management and finite natural resources serving an expanding world population - these reasons mean that urgent action is required to transition to solutions which minimize environmental impact and are sustainable. We are at the start of the low - carbon revolution and those that have started on their low - carbon journey already are seeing benefits such as new markets and customers, improved economic, social and environmental performance, and reduced bills and risks. Green investment banks offer alternative financial services: green car loans, energy efficiency mortgages, alternative energy venture capital, eco - savings deposits and green credit cards. These items represent innovative financial products.

  6. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

    Directory of Open Access Journals (Sweden)

    Michelle de Campos Soriani Azevedo

    2017-01-01

    Full Text Available Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR, a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV and negative (NPV predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H, the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

  7. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.

    Science.gov (United States)

    Azevedo, Michelle de Campos Soriani; Ramuno, Natália Mortari; Fachin, Luciana Raquel Vincenzi; Tassa, Mônica; Rosa, Patrícia Sammarco; Belone, Andrea de Faria Fernandes; Diório, Suzana Madeira; Soares, Cleverson Teixeira; Garlet, Gustavo Pompermaier; Trombone, Ana Paula Favaro

    Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  8. Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

    Directory of Open Access Journals (Sweden)

    Sandra Christine Andersen

    2017-11-01

    Full Text Available In microbial food safety, molecular methods such as quantitative PCR (qPCR and next-generation sequencing (NGS of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked with 103 or 106 colony-forming units (CFU/g Campylobacter jejuni, as well as porcine fecal samples spiked with 103 or 106 CFU/g Salmonella typhimurium. DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA fragmentation during library preparation, amount of DNA available for sequencing, amount of sequencing data, distribution of data between samples in a batch, and data insert size; none showed any correlation with the target ratio of the spiking organism detected in sequencing data. Surprisingly, diagnostic metagenomics can have better detection sensitivity than qPCR for samples spiked with 103 CFU/g C. jejuni. The study also showed that qPCR and sequencing results may be different due to inhibition in one of the methods. In conclusion, qPCR cannot uncritically be used as an indicator for the optimization of sample preparation for diagnostic metagenomics.

  9. Green Power Partnership Videos

    Science.gov (United States)

    The Green Power Partnership develops videos on a regular basis that explore a variety of topics including, Green Power partnership, green power purchasing, Renewable energy certificates, among others.

  10. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Directory of Open Access Journals (Sweden)

    David E Lucero

    2014-12-01

    Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  11. Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches.

    Science.gov (United States)

    Schmidt, Gunilla Veslemøy; Mellerup, Anders; Christiansen, Lasse Engbo; Ståhl, Marie; Olsen, John Elmerdahl; Angen, Øystein

    2015-01-01

    The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.

  12. Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

    Science.gov (United States)

    Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

    2014-01-01

    Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

  13. Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

    Science.gov (United States)

    Lucero, David E; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W; Peña, Reynaldo; Morrissey, Leslie A; Rizzo, Donna M; Stevens, Lori

    2014-12-01

    In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

  14. Pre-amplification in the context of high-throughput qPCR gene expression experiment

    Czech Academy of Sciences Publication Activity Database

    Korenková, Vlasta; Scott, J.; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjoback, R.

    2015-01-01

    Roč. 16, č. 5 (2015) ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP304/12/1585; GA ČR(CZ) GA15-08239S; GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : High-throughput qPCR * Gene expression * Exponential pre-amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2015

  15. Going Green

    Science.gov (United States)

    Witkowsky, Kathy

    2009-01-01

    Going green saves money and can even make money. Sustainable practices promote better health, less absenteeism, and more productivity. They also attract students, who are paying increasing attention to schools' environmental policies. Beyond being the smart thing to do, administrators at the University of Washington say repeatedly, it's the right…

  16. Going Green

    Centers for Disease Control (CDC) Podcasts

    2008-04-18

    This podcast is for a general audience and provides information on how to recycle, re-use, and restore. It also covers the benefits of “Going Green" on the environment, health, and social interaction.  Created: 4/18/2008 by National Center for Environmental Health (NCEH), ATSDR.   Date Released: 5/8/2008.

  17. Buying Green

    Science.gov (United States)

    Layng, T. V. Joe

    2010-01-01

    In "Buying Green," Joe Layng recognizes that, like all choices we make, our decisions as consumers are more likely to be influenced by their short-term consequences for us as individuals (price, quality) than they are by their long-term consequences for society (environmental impact). He believes that the equation can be tilted in favor of greener…

  18. Automatically Green

    DEFF Research Database (Denmark)

    Sunstein, Cass R.; Reisch, Lucia

    2014-01-01

    reasons include the power of suggestion; inertia and procrastination; and loss aversion. If well-chosen, green defaults are likely to have large effects in reducing the economic and environmental harms associated with various products and activities. Such defaults may or may not be more expensive...

  19. Automatically Green

    DEFF Research Database (Denmark)

    Sunstein, Cass R.; Reisch, Lucia

    reasons include the power of suggestion; inertia and procrastination; and loss aversion. If well-chosen, green defaults are likely to have large effects in reducing the economic and environmental harms associated with various products and activities. Such defaults may or may not be more expensive...

  20. Green pioneers.

    Science.gov (United States)

    Trueland, Jennifer

    The government has set tough targets for the NHS in England to reduce its carbon footprint. In this article, nurses and managers at Nottinghamshire Healthcare NHS Trust explain how a programme of 'greening' initiatives - including a trial of electric cars for community staff - have slashed the trust's CO2 output.

  1. Greening infrastructure

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2014-10-01

    Full Text Available , and coal, with a concomitant release of greenhouse gases. Green infrastructure seeks to perform those functions in a manner that, at the very least, minimises its impact on the natural environment and, at best, enhances the quality of the natural...

  2. Q-PCR Based Culture-Independent Enumeration and Detection of Enterobacter: An Emerging Environmental Human Pathogen in Riverine Systems and Potable Water

    Science.gov (United States)

    Patel, Chandra B.; Shanker, Rishi; Gupta, Vijai K.; Upadhyay, Ram S.

    2016-01-01

    The availability of safe and pristine water is a global challenge when large numbers of natural and anthropogenic water resources are being depleted with faster rate. The remaining water resources are severely contaminated with various kinds of contaminants including microorganisms. Enterobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. Enterobacter was earlier used as an indicator bacterium along with other fecal Coliforms namely Escherichia coli, Citrobacter, and Klebsiella, but it is now known to cause various diseases in human beings. In this study, we have collected 55 samples from potable water and riverine system and proved their presence using their conserved sequences of 16S rRNA and 23S rRNA genes with the help of SYBR green real-time PCR, which showed very high specificity for the detection of Enterobacter. The Enterobacter counts in potable water were found to 1290 ± 32.89 to 1460 ± 39.42 cfu/100 ml. The Enterobacter levels in surface water were 1.76 × 104 ± 492, 1.33 × 104 ± 334, 1.15 × 104 ± 308, 2.56 × 104 ± 802, 2.89 × 104 ± 962, 8.16 × 104 ± 3443 cfu/100 ml; the levels of Enterobacter contamination associated with hydrophytes were 4.80 × 104 ± 1804, 3.48 × 104 ± 856, 8.50 × 104 ± 2074, 8.09 × 104 ± 1724, 6.30 × 104 ± 1738, 3.68 × 104 ± 949 cfu/10 g and the Enterobacter counts in sediments of the river, were 2.36 × 104 ± 703, 1.98 × 104 ± 530, 9.92 × 104 ± 3839, 6.80 × 104 ± 2230, 8.76 × 104 ± 3066 and 2.34 × 104 ± 732 cfu/10 g at the sampling Site #1, Site #2, Site #3, Site #4, Site #5, and Site #6, respectively. The assay could be used for the regular monitoring of potable water and other water reservoirs to check waterborne outbreaks. PMID:26925044

  3. Q-PCR based culture-independent enumeration and detection of Enterobacter: an emerging environmental human pathogen in riverine systems and potable water

    Directory of Open Access Journals (Sweden)

    Ram Sanmukh Upadhyay

    2016-02-01

    Full Text Available The availability of safe and pristine water is a global challenge when large numbers of natural and anthropogenic water resources are being depleted with faster rate. The remaining water resources are severely contaminated with various kinds of contaminants including microorganisms. Enterobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. Enterobacter was earlier used as an indicator bacterium along with other fecal Coliforms namely Escherichia coli, Citrobacter and Klebsiella, but it is now known to cause various diseases in human beings. In this study, we have collected 55 samples from potable water and riverine system and proved their presence using their conserved sequences of 16S rRNA and 23S rRNA genes with the help of SYBR green real-time PCR, which showed very high specificity for the detection of Enterobacter. The Enterobacter counts in potable water were found to 1290 ± 32.89 to 1460 ± 39.42 cfu/100ml. The Enterobacter levels in surface water were 1.76 x104 ±492, 1.33 x104 ±334, 1.15 x104 ±308, 2.56 x104 ±802, 2.89 x104 ±962, 8.16 x104 ±3443 cfu/100ml; the levels of Enterobacter contamination associated with hydrophytes were 4.80 x104 ±1804, 3.48 x104 ±856, 8.50 x104 ±2074, 8.09 x104 ±1724, 6.30 x104 ±1738, 3.68 x104 ±949 cfu/10 g and the Enterobacter counts in sediments of the river, were 2.36 x104 ±703, 1.98 x104 ±530, 9.92 x104 ±3839, 6.80 x104 ±2230, 8.76 x104 ±3066 and 2.34 x104 ±732 cfu/10g at the sampling Site #1, Site # 2, Site # 3, Site # 4, Site # 5 and Site # 6 respectively. The assay could be used for the regular monitoring of potable water and other water reservoirs to check waterborne outbreaks.

  4. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Directory of Open Access Journals (Sweden)

    Haug Trude M

    2010-11-01

    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  5. QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology.

    Science.gov (United States)

    Meyer, Joel N

    2010-04-01

    The quantitative PCR (QPCR) assay for DNA damage and repair has been used extensively in laboratory species. More recently, it has been adapted to ecological settings. The purpose of this article is to provide a detailed methodological guide that will facilitate its adaptation to additional species, highlight its potential for ecotoxicological and biomonitoring work, and critically review the strengths and limitations of this assay. Major strengths of the assay include very low (nanogram to picogram) amounts of input DNA; direct comparison of damage and repair in the nuclear and mitochondrial genomes, and different parts of the nuclear genome; detection of a wide range of types of DNA damage; very good reproducibility and quantification; applicability to properly preserved frozen samples; simultaneous monitoring of relative mitochondrial genome copy number; and easy adaptation to most species. Potential limitations include the limit of detection (approximately 1 lesion per 10(5) bases); the inability to distinguish different types of DNA damage; and the need to base quantification of damage on a control or reference sample. I suggest that the QPCR assay is particularly powerful for some ecotoxicological studies.

  6. Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

    Directory of Open Access Journals (Sweden)

    Ana Beatriz F. Pacheco

    2016-06-01

    Full Text Available The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR. Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk.

  7. Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

    Directory of Open Access Journals (Sweden)

    Genevieve Housman

    2015-11-01

    Full Text Available Zoonotic pathogens that cause leprosy (Mycobacterium leprae and tuberculosis (Mycobacterium tuberculosis complex, MTBC continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

  8. Development of a qPCR method to rapidly assess the function of NKT cells.

    Science.gov (United States)

    Sohn, Silke; Tiper, Irina; Japp, Emily; Sun, Wenji; Tkaczuk, Katherine; Webb, Tonya J

    2014-05-01

    NKT cells comprise a rare, but important subset of T cells which account for ~0.2% of the total circulating T cell population. NKT cells are known to have anti-tumor functions and rapidly produce high levels of cytokines following activation. Several clinical trials have sought to exploit the effector functions of NKT cells. While some studies have shown promise, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender, thus limiting their therapeutic efficacy. These studies indicate that baseline levels of activation should be assessed before initiating an NKT cell based immunotherapeutic strategy. The goal of this study was to develop a sensitive method to rapidly assess NKT cell function. We utilized artificial antigen presenting cells in combination with qPCR in order to determine NKT cell function in peripheral blood mononuclear cells from healthy donors and breast cancer patients. We found that NKT cell activation can be detected by qPCR, but not by ELISA, in healthy donors as well as in breast cancer patients following four hour stimulation. This method utilizing CD1d-expressing aAPCs will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Novel qPCR systems for olive (Olea europaea L.) authentication in oils and food.

    Science.gov (United States)

    Ramos-Gómez, Sonia; Busto, María D; Albillos, Silvia M; Ortega, Natividad

    2016-03-01

    The traceability of olive oil is an unresolved issue that remains a challenge. In this field, DNA-based techniques are very powerful tools for discrimination that are less negatively influenced by environmental conditions than other techniques. More specifically, quantitative real time PCR (qPCR) achieves a high degree of sensitivity, although the DNA that it can directly isolate from these oils presents drawbacks. Our study reports the analysis of eight systems, in order to determine their suitability for olive detection in oil and oil-derived foodstuffs. The eight systems were analyzed on the basis of their sensitivity and specificity in the qPCR assay, their relative sensitivity to olive DNA detection and DNA mixtures, their sensitivity and specificity to olive in vegetable oils and the detection of olive in commercial products. The results show that the PetN-PsbM system, designed in this study, is a suitable and reliable technique in relation to olive oil and olive ingredients in both food authentication and food safety processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Pengaruh Green Marketing Hotel Terhadap Green Consumer Behavior

    OpenAIRE

    Yo Fernandez, Eunike Christe; Tjoanda, Evelyn

    2017-01-01

    Penelitian ini dilakukan untuk mengetahui pengaruh dari green marketing hotel terhadap green consumer behavior. Green marketing memiliki 3 dimensi, yaitu green product, green price, dan green promotion. Penelitian ini melibatkan 272 responden masyarakat Surabaya dan menggunakan metode regresi linear berganda. Hasil penelitian menunjukkan bahwa green product dan green price berpengaruh secara positif dan signifikan sedangkan green promotion berpengaruh namun tidak signifikan terhadap green con...

  11. Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

    Science.gov (United States)

    Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T

    2015-12-01

    To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

  12. From green architecture to architectural green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    of green architecture. The paper argues that this greenification of facades is insufficient. The green is only a skin cladding the exterior envelope without having a spatial significance. Through the paper it is proposed to flip the order of words from green architecture to architectural green....... Architectural green could signify green architecture with inclusive interrelations between green and space, built and unbuilt, inside and outside. The aim of the term is to reflect a new focus in green architecture – its architectural performance. Ecological issues are not underestimated or ignored, but so far...... they have overshadowed the architectural potential of green architecture. The paper questions how a green space should perform, look like and function. Two examples are chosen to demonstrate thorough integrations between green and space. The examples are public buildings categorized as pavilions. One...

  13. Green Computing

    Directory of Open Access Journals (Sweden)

    K. Shalini

    2013-01-01

    Full Text Available Green computing is all about using computers in a smarter and eco-friendly way. It is the environmentally responsible use of computers and related resources which includes the implementation of energy-efficient central processing units, servers and peripherals as well as reduced resource consumption and proper disposal of electronic waste .Computers certainly make up a large part of many people lives and traditionally are extremely damaging to the environment. Manufacturers of computer and its parts have been espousing the green cause to help protect environment from computers and electronic waste in any way.Research continues into key areas such as making the use of computers as energy-efficient as Possible, and designing algorithms and systems for efficiency-related computer technologies.

  14. Green Gold

    International Nuclear Information System (INIS)

    Salamandra Martinez, Carlos

    2004-01-01

    The main purpose of this work is to offer a general panoramic of the processes or experiences pilot that are carried out in the Project Green Gold, as strategy of environmental sustainability and organizational invigoration in Choco, especially in the 12 communities of the municipalities of Tado and Condoto. It is also sought to offer a minimum of information on the techniques of handmade production and to show the possibilities to carry out in a rational way the use and use of the natural resources. The Project Green Gold is carried out by the Corporation Green Gold (COV) and co-financed with resources of international and national character, the intervention of the financial resources it achievement mainly for the use of clean processes in the extraction stages and metals benefit. The project is centered primarily in the absence of use of products or toxic substances as the mercury, fair trade, organizational invigoration, execution of 11 approaches and certification of the metals Gold and Platinum. The COV, it has come executing the proposal from the year 2001 with the premise of contributing to the balance between the rational exploitation of the natural resources and the conservation of the environment in the Choco. In the project they are used technical handmade characteristic of the region framed inside the mining activity and production activities are diversified in the productive family units. Those producing with the support of entities of juridical character, specify the necessary game rules for the extraction and products commercialization

  15. Greens of the European Green Capitals

    Science.gov (United States)

    Cömertler, Seval

    2017-10-01

    Well established and maintained green areas have a key role on reaching the high quality of life and sustainability in urban environments. Therefore, green areas must be carefully accounted and evaluated in the urban planning affairs. In this context, the European Green Capitals, which attach a great importance to the green areas, have a great potential to act as a role model for both small and big cities in all around the world. These leading cities (chronologically, Stockholm, Hamburg, Vitoria-Gasteiz, Nantes, Copenhagen, Bristol, Ljubljana, Essen and Nijmegen) are inspiring for the other cities which seek to achieve more sustainable and environmentally friendly places through green areas. From this point of view, the aim of this paper was to investigate the green areas of the European Green Capitals. The paper covered whole European Green Capitals, and the application form of each Green Capital was used as a primary data source. Consequently, the paper put forwarded that the European Green Capitals have considerably large amount and high proportion of green areas. Further, these cities provide an excellent access to the public green areas. As a result of abundant provision and proper distribution, the almost all citizens in most of the Green Capitals live within a distance of 300 meters to a green area. For further researches, the paper suggested that these green capitals should be investigated in terms of their efforts, measures, goals and plans, policies and implications to administer, to protect, to enhance and to expand the green areas.

  16. Relationship and variation of qPCR and culturable enterococci estimates in ambient surface waters are predictable

    Science.gov (United States)

    Whitman, Richard L.; Ge, Zhongfu; Nevers, Meredith B.; Boehm, Alexandria B.; Chern, Eunice C.; Haugland, Richard A.; Lukasik, Ashley M.; Molina, Marirosa; Przybyla-Kelly, Kasia; Shively, Dawn A.; White, Emily M.; Zepp, Richard G.; Byappanahalli, Muruleedhara N.

    2010-01-01

    The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P 10CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

  17. From green architecture to architectural green

    DEFF Research Database (Denmark)

    Earon, Ofri

    2011-01-01

    of green architecture. The paper argues that this greenification of facades is insufficient. The green is only a skin cladding the exterior envelope without having a spatial significance. Through the paper it is proposed to flip the order of words from green architecture to architectural green...... that describes the architectural exclusivity of this particular architecture genre. The adjective green expresses architectural qualities differentiating green architecture from none-green architecture. Currently, adding trees and vegetation to the building’s facade is the main architectural characteristics...

  18. Image data analysis in qPCR: A method for smart analysis of DNA amplification

    Directory of Open Access Journals (Sweden)

    Massimo Orazio Spata

    2015-12-01

    Full Text Available In this paper, a method for the direct quantitative analysis of amplified DNA via q-Polymerase Chain Reaction (qPCR in miniaturised silicon-based chip system is presented. The designed tool presented here allows the automatic extraction of meaningful information from input fluorescent images by means of digital image processing algorithm. In particular, a smart mathematical model, optimizing the integration of all the analysis steps of the fluorescence data from on chip multiple real time PCR, is described. Such a tool is able to load the digital input images, select and smartly detect the region of interest for fluorescence, elaborate the data input, calculate the average fluorescence values and finally, plot the fitted curve as output, giving also, for each well, both the Cycle Threshold (CT and Slope parameters.

  19. Real-time pcr (qpcr) assay for rhizoctonia solani anastomoses group ag2-2 iiib

    International Nuclear Information System (INIS)

    Abbas, S.J.; Ahmad, B.

    2014-01-01

    Rhizoctonia solani anastomosis group AG2-2 IIIB is a severe sugar beet and maize pathogen. It causes crown and root rot disease which leads to yield losses world-wide. The soil-borne pathogen is difficult to detect and quantify by conventional methods. We developed a real-time PCR (qPCR) assay for the quantification of genomic DNA of Rhizoctonia solani AG2-2 IIIB based on the ITS region of rDNA genes. The limit of quantification of the assay is 1.8 pg genomic DNA. The amplification efficiency was 96.4. The assay will be helpful in the diagnoses of Rhizoctonia solani infection of sugar beet and maize roots and in the quantification of R. solani AG2-2 IIIB inoculum in plant debris and soil. (author)

  20. Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

    Science.gov (United States)

    Eberle, Kirsten C.; Venn-Watson, Stephanie K.; Jensen, Eric D.; Porter, Tracy J.; Waters, Theresa E.; Sacco, Randy E.

    2017-01-01

    Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus) health. Quantitative PCR (qPCR) can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC). This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL) and concanavalin A (1 μg/mL) for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α). Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies. PMID:29272269

  1. Molecular Quantification of Zooplankton Gut Content: The Case For qPCR

    Science.gov (United States)

    Frischer, M. E.; Walters, T. L.; Gibson, D. M.; Nejstgaard, J. C.; Troedsson, C.

    2016-02-01

    The ability to obtain information about feeding selectivity and rates in situ for zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, directly estimating feeding selection and rates of zooplankton, without bias, associated with culturing conditions has been notoriously difficult. A potential approach for addressing this problem is to target prey-specific DNA as a marker for prey ingestion and selection. In this study we report the development of a differential length amplification quantitative PCR (dla-qPCR) assay targeting the 18S rRNA gene to validate the use of a DNA-based approach to quantify consumption of specific plankton prey by the pelagic tunicate (doliolid) Dolioletta gegenbauri. Compared to copepods and other marine animals, the digestion of prey genomic DNA inside the gut of doliolids is low. This method minimizes potential underestimations, and therefore allows prey DNA to be used as an effective indicator of prey consumption. We also present an initial application of a qPCR-assay to estimate consumption of specific prey species on the southeastern continental shelf of the U.S., where doliolids stochastically bloom in response to upwelling events. Estimated feeding rates, based on qPCR, were in the same range as those estimated from clearance rates in laboratory feeding studies. In the field, consumption of specific prey, including the centric diatom Thalassiosira spp. was detected in the gut of wild caught D. gegenbauri at the levels consistent with their abundance in the water column at the time of collection. Thus, both experimental and field investigations support the hypothesis that a qPCR approach will be useful for the quantitative investigation of the in situ diet of D. gegenbauri without introduced bias' associated with cultivation.

  2. Monitoring bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR.

    Directory of Open Access Journals (Sweden)

    Amelia Ruth Hofstetter

    Full Text Available Both veterinarians caring for dolphins in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin (Tursiops truncatus health. Quantitative PCR (qPCR can be used to assess cytokine transcription patterns of peripheral blood mononuclear cells (PBMC. This can supplement currently available blood tests with information on immune status. Full realization of this potential requires establishment of normal ranges of cytokine gene transcription levels in bottlenose dolphins. We surveyed four dolphins over the span of seven months by serial bleeds. PBMC were stimulated with phytohaemagglutinin (1, 5, and 10 μg/mL and concanavalin A (1 μg/mL for 48 H in vitro. RNA from these cultures was probed by qPCR using Tursiops truncatus-specific primers (IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IL-18, IFN-γ and TNF-α. Two blood samples from an additional bottlenose dolphin diagnosed with acute pulmonary disease add further perspective to the data. We observed that mitogen choice made a significant difference in the magnitude of gene transcription observed. On the other hand, most cytokines tested exhibited limited intra-animal variation. However, IL-6 and IL-12p40 differed between older and younger dolphins. Furthermore, the magnitude of mitogenic response clusters the tested cytokines into three groups. The data provide a reference for the selection of target cytokine mRNAs and their expected range of mitogen-stimulated cytokine gene transcription for future studies.

  3. Green roofs

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2010-04-01

    Full Text Available for green roofs. A typical method makes use of a studded membrane (resembling an egg box) laid on a suitable waterproofing which, together, provides the waterproofing and the water drainage. A Geotextile blanket is then laid on top of the studded... be advisable to lay a supporting layer between the insulation and the studded membrane to protect the insulation. A further variation is to replace the studded membrane with a gravel drainage layer followed by the growing medium. In this instance it would...

  4. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    Science.gov (United States)

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans.

    Directory of Open Access Journals (Sweden)

    Fernanda Gutierrez-Rodrigues

    Full Text Available Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH, and quantitative PCR (qPCR. Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2 = 0.60; p<0.0001 and patients (R(2 = 0.51; p<0.0001. In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2 = 0.35; p<0.0001 and low correlation for patients (R(2 = 0.20; p = 0.001; Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2 = 0.33; p<0.0001 and did not correlate in the comparison of patients' samples (R(2 = 0.1, p = 0.08. Intra-assay coefficient of variation (CV was similar for flow-FISH (10.8 ± 7.1% and qPCR (9.5 ± 7.4%; p = 0.35, but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02. Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100% and specific (93% and 89%, respectively to distinguish very short telomeres. However, qPCR sensitivity (40% and specificity (63% to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity. In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human

  6. Green shipping management

    CERN Document Server

    Lun, Y H Venus; Wong, Christina W Y; Cheng, T C E

    2016-01-01

    This book presents theory-driven discussion on the link between implementing green shipping practices (GSP) and shipping firm performance. It examines the shipping industry’s challenge of supporting economic growth while enhancing environmental performance. Consisting of nine chapters, the book covers topics such as the conceptualization of green shipping practices (GSPs), measurement scales for evaluating GSP implementation, greening capability, greening and performance relativity (GPR), green management practice, green shipping network, greening capacity, and greening propensity. In view of the increasing quest for environment protection in the shipping sector, this book provides a good reference for firms to understand and evaluate their capability in carrying out green operations on their shipping activities.

  7. Green chemistry

    International Nuclear Information System (INIS)

    Warner, John C.; Cannon, Amy S.; Dye, Kevin M.

    2004-01-01

    A grand challenge facing government, industry, and academia in the relationship of our technological society to the environment is reinventing the use of materials. To address this challenge, collaboration from an interdisciplinary group of stakeholders will be necessary. Traditionally, the approach to risk management of materials and chemicals has been through inerventions intended to reduce exposure to materials that are hazardous to health and the environment. In 1990, the Pollution Prevention Act encouraged a new tact-elimination of hazards at the source. An emerging approach to this grand challenge seeks to embed the diverse set of environmental perspectives and interests in the everyday practice of the people most responsible for using and creating new materials--chemists. The approach, which has come to be known as Green Chemistry, intends to eliminate intrinsic hazard itself, rather than focusing on reducing risk by minimizing exposure. This chapter addresses the representation of downstream environmental stakeholder interests in the upstream everyday practice that is reinventing chemistry and its material inputs, products, and waste as described in the '12 Principles of Green Chemistry'

  8. Green chemistry

    International Nuclear Information System (INIS)

    Colonna, P.

    2006-01-01

    The depletion of world fossil fuel reserves and the involvement of greenhouse gases in the global warming has led to change the industrial and energy policies of most developed countries. The goal is now to reserve petroleum to the uses where it cannot be substituted, to implement renewable raw materials obtained from plants cultivation, and to consider the biodegradability of molecules and of manufactured objects by integrating the lifetime concept in their expected cycle of use. The green chemistry includes the design, development and elaboration of chemical products and processes with the aim of reducing or eliminating the use and generation of harmful compounds for the health and the environment, by adapting the present day operation modes of the chemical industry to the larger framework of the sustainable development. In addition to biofuels, this book reviews the applications of green chemistry in the different industrial processes in concern. Part 1 presents the diversity of the molecules coming from renewable carbon, in particular lignocellulose and the biotechnological processes. Part 2 is devoted to materials and treats of the overall available technological solutions. Part 3 focusses on functional molecules and chemical intermediates, in particular in sugar- and fats-chemistry. Part 4 treats of biofuels under the aspects of their production and use in today's technologies. The last part deals with the global approaches at the environmental and agricultural levels. (J.S.)

  9. Green urbanity

    Directory of Open Access Journals (Sweden)

    Alenka Fikfak

    2012-01-01

    Full Text Available Tourism and other culture-based types of small business, which are the leitmotif in the planning of the Europark Ruardi, are becoming the guiding motif in the spatial development of urban centres that are influenced by dynamic transformation processes. The system should build upon the exploitation of both local and regional environmental features. This would encourage the quest for special environmental features, with an emphasis on their conservation, i.e. sustainable development, and connections in a wider context.The Europark is seen as a new strategic point of the Zasavje Region (the region of the central Sava Valley, which is linked to other important points in a region relevant for tourism. Due to the "smallness" of the region and/or the proximity of such points, development can be fast and effective. The interaction of different activities in space yields endless opportunities for users, who choose their own goals and priorities in the use of space. Four theme areas of the Europark area planning are envisaged. The organisation of activities is based on the composition of the mosaic field patterns, where green fields intertwine with areas of different, existing and new, urban functions. The fields of urban and recreation programmes are connected with a network of green areas and walking trails, along which theme park settings are arranged.

  10. Green Manufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Patten, John

    2013-12-31

    Green Manufacturing Initiative (GMI): The initiative provides a conduit between the university and industry to facilitate cooperative research programs of mutual interest to support green (sustainable) goals and efforts. In addition to the operational savings that greener practices can bring, emerging market demands and governmental regulations are making the move to sustainable manufacturing a necessity for success. The funding supports collaborative activities among universities such as the University of Michigan, Michigan State University and Purdue University and among 40 companies to enhance economic and workforce development and provide the potential of technology transfer. WMU participants in the GMI activities included 20 faculty, over 25 students and many staff from across the College of Engineering and Applied Sciences; the College of Arts and Sciences' departments of Chemistry, Physics, Biology and Geology; the College of Business; the Environmental Research Institute; and the Environmental Studies Program. Many outside organizations also contribute to the GMI's success, including Southwest Michigan First; The Right Place of Grand Rapids, MI; Michigan Department of Environmental Quality; the Michigan Department of Energy, Labor and Economic Growth; and the Michigan Manufacturers Technical Center.

  11. Green Transformational Leadership and Green Performance: The Mediation Effects of Green Mindfulness and Green Self-Efficacy

    Directory of Open Access Journals (Sweden)

    Yu-Shan Chen

    2014-09-01

    Full Text Available No prior literature explores the influence of green transformational leadership on green performance, thus, this study develops a novel research framework to fill the research gap. This study investigates the influence of green transformational leadership on green performance and discusses the mediation effects of green mindfulness and green self-efficacy by means of structural equation modeling (SEM. The results indicate that green transformational leadership positively influences green mindfulness, green self-efficacy, and green performance. Moreover, this study demonstrates that the positive relationship between green transformational leadership and green performance is partially mediated by the two mediators: green mindfulness and green self-efficacy. It means that green transformational leadership can not only directly affect green performance positively but also indirectly affect it positively through green mindfulness and green self-efficacy. Therefore, firms need to raise their green transformational leadership, green mindfulness, and green self-efficacy to increase their green performance.

  12. Green Power Partner Resources

    Science.gov (United States)

    EPA Green Power Partners can access tools and resources to help promote their green power commitments. Partners use these tools to communicate the benefits of their green power use to their customers, stakeholders, and the general public.

  13. Green business will remain green

    International Nuclear Information System (INIS)

    Marcan, P.

    2008-01-01

    It all started with two words. Climate change. The carbon dioxide trading scheme, which was the politicians' idea on solving the number one global problem, followed. Four years ago, when the project was begun, there was no data for project initiation. Quotas for polluters mainly from energy production and other energy demanding industries were distributed based on spreadsheets, maximum output and expected future development of economies. Slovak companies have had a chance to profit from these arrangements since 2005. Many of them took advantage of the situation and turned the excessive quotas into an extraordinary profit which often reached hundreds of million Sk. The fact that the price of free quotas offered for sale dropped basically to 0 in 2006 only proved that the initial distribution was too generous. And the market reacted to the first official measurements of emissions. Slovak companies also contributed to this development. However, when planning the maximum emission volumes for 2008-2012 period, in spite of the fact that actual data were available, their expectations were not realistic. A glance at the figures in the proposal of the Ministry of Environment is sufficient to realize that there will be no major change in the future. And so for many Slovak companies business with a green future will remain green for the next five years. The state decided to give to selected companies even more free space as far as emissions are concerned. The most privileged companies can expect quotas increased by tens of percent. (author)

  14. An easy 'one tube' method to estimate viability of Cryptosporidium oocysts using real-time qPCR

    NARCIS (Netherlands)

    Paziewska-Harris, A.; Schoone, G.; Schallig, H. D. F. H.

    2016-01-01

    Viability estimation of the highly resistant oocysts of Cryptosporidium remains a key issue for the monitoring and control of this pathogen. We present here a simple 'one tube' quantitative PCR (qPCR) protocol for viability estimation using a DNA extraction protocol which preferentially solubilizes

  15. Comparison of Enterococcus qPCR analysis results from fresh and marine waters on two real-tme instruments

    Science.gov (United States)

    The U.S. Environmental Protection Agency (EPA) will be recommending a quantitativ e polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this o...

  16. A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir; Krych, Lukasz; Nielsen, Dennis Sandris

    2017-01-01

    Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows...

  17. Gut Low Density Array (GULDA), a novel qPCR approach to the study of the intestinal microbial ecosystem

    DEFF Research Database (Denmark)

    Bergström, Anders; Andersen, Jens Bo; Licht, Tine Rask

    developed GULDA; a qPCR low-density array with particular focus on bacteria of relevance to the human gut microbiota. The output is given as arbitrary bacterial quantities, which for large sample numbers allow for further characterization of the gut microbiota by uni- and multivariate statistical methods....

  18. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

    Science.gov (United States)

    The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay rather than the entire sample process. Our objective was to develop a method to determine the 95% LOD (lowest co...

  19. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree

    2012-09-23

    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  20. Central Region Green Infrastructure

    Data.gov (United States)

    Minnesota Department of Natural Resources — This Green Infrastructure data is comprised of 3 similar ecological corridor data layers ? Metro Conservation Corridors, green infrastructure analysis in counties...

  1. Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles

    Directory of Open Access Journals (Sweden)

    Anna Kłopot

    2017-11-01

    Full Text Available The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay, but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between “microbiological disappearance” and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or

  2. Cryptosporidium diagnosis by qPCR in cats at Rio de Janeiro state, Brazil

    Directory of Open Access Journals (Sweden)

    Lara Patrícia Santos Carrasco

    2016-11-01

    Full Text Available ABSTRACT. Carrasco L.P.S., Oliveira R.L.S., Moreira C.M.R., Santos C.R.G.R., Corgozinho K.B. & Souza H.J.M. [Cryptosporidium diagnosis by qPCR in cats at Rio de Janeiro state, Brazil.] Diagnóstico de Cryptosporidium spp. pela técnica de qPCR em gatos no estado do Rio de Janeiro, Brasil. Revista Brasileira de Medicina Veterinária, 38(Supl.:22-26, 2016. Programa de Pós-Graduação em MedicinaVeteriná- ria, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR-465, Km 7, Seropédica, RJ 23890-000, Brasil. E-mail: carrasco.lara@gmail.com Cryptosporidium spp. is recognized as an important etiologic agent of diarrhea in many countries. The aim of this study was to detect the presence of DNA of the parasite Cryptosporidium spp. in feces of cats with history of chronic diarrhea attended in the Feline Medicine Sector of the Veterinary Hospital of the Federal Rural University of Rio de Janeiro, by the polymerase chain reaction technique in real time (RT-PCR. In this study, 100 animals were admitted, of any breed or sex and from 8 weeks of age. As inclusion criteria, patients had to have diarrhea history for more than three weeks, with little success of clinical response to previously established therapies. From the samples obtained by collecting via washing the animal colon and spontaneous defecation, methods of direct examination of the feces, centrifugal flotation technique and real-time PCR were carried out. Of all cats selected for this study, 10% showed infection by Cryptosporidium spp. Most positive animals were aged over one year (70% and only 30% had up to one year old. Cats were 50% purebred and 50% were domestic short hair cats. The clinical signs presented by these cats at the time of consultation were diarrhea (60% and prolapsed rectum (40%. Four animals had co-infections with other enteropathogens (40%, such as Giardia, Toxocara sp. or Tritrichomonas fetus alone or combined. We concluded that infection by

  3. The green building envelope : Vertical greening

    NARCIS (Netherlands)

    Ottelé, M.

    2011-01-01

    Planting on roofs and façades is one of the most innovative and fastest developing fields of green technologies with respect to the built environment and horticulture. This thesis is focused on vertical greening of structures and to the multi-scale benefits of vegetation. Vertical green can improve

  4. The Determinants of Green Radical and Incremental Innovation Performance: Green Shared Vision, Green Absorptive Capacity, and Green Organizational Ambidexterity

    Directory of Open Access Journals (Sweden)

    Yu-Shan Chen

    2014-11-01

    Full Text Available This study proposes a new concept, green organisational ambidexterity, that integrates green exploration learning and green exploitation learning simultaneously. Besides, this study argues that the antecedents of green organisational ambidexterity are green shared vision and green absorptive capacity and its consequents are green radical innovation performance and green incremental innovation performance. The results demonstrate that green exploration learning partially mediates the positive relationships between green radical innovation performance and its two antecedents—green shared vision and green absorptive capacity. In addition, this study indicates that green exploitation learning partially mediates the positive relationships between green incremental innovation performance and its two antecedents—green shared vision and green absorptive capacity. Hence, firms have to increase their green shared vision, green absorptive capacity, and green organisational ambidexterity to raise their green radical innovation performance and green incremental innovation performance.

  5. Therapeutic and Imaging Applications of Dopamine Receptors in Breast Cancer

    Science.gov (United States)

    2015-09-01

    Applied Biosystems, Carlsbad, CA, USA). Product purity was verified using DNA melting curve analysis and agarose gel electrophoresis . PCR efficiency... agarose gel and photographed. Quantitative real-time PCR (qPCR) was done using SYBR Green, and products were detected with a StepOnePlus instrument...which can be leveraged, through the use of D1R agonists, as a novel therapy for suppressing tumor growth and reducing resistance to chemotherapy

  6. How Green is 'Green' Energy?

    Science.gov (United States)

    Gibson, Luke; Wilman, Elspeth N; Laurance, William F

    2017-12-01

    Renewable energy is an important piece of the puzzle in meeting growing energy demands and mitigating climate change, but the potentially adverse effects of such technologies are often overlooked. Given that climate and ecology are inextricably linked, assessing the effects of energy technologies requires one to consider their full suite of global environmental concerns. We review here the ecological impacts of three major types of renewable energy - hydro, solar, and wind energy - and highlight some strategies for mitigating their negative effects. All three types can have significant environmental consequences in certain contexts. Wind power has the fewest and most easily mitigated impacts; solar energy is comparably benign if designed and managed carefully. Hydropower clearly has the greatest risks, particularly in certain ecological and geographical settings. More research is needed to assess the environmental impacts of these 'green' energy technologies, given that all are rapidly expanding globally. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

    Directory of Open Access Journals (Sweden)

    Lindsey M. Skrdlant

    2018-03-01

    Full Text Available Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G] for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations.

  8. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    Science.gov (United States)

    Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

    2012-01-01

    Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix. PMID:23105972

  9. A multimarker qPCR platform for the characterisation of endometrial cancer.

    Science.gov (United States)

    Supernat, Anna; Łapińska-Szumczyk, Sylwia; Majewska, Hanna; Gulczyński, Jacek; Biernat, Wojciech; Wydra, Dariusz; Zaczek, Anna J

    2014-02-01

    The molecular background of endometrial cancer (EC) has not been fully elucidated. In the present study, we developed a quantitative PCR (qPCR) platform to examine the gene dosages of the potential molecular markers MGB1, TOP2A, ERBB1-4, MYC, CCND1, ESR1 and PI3K. The platform was applied in samples collected from 157 EC patients (stage I-IV) to verify its clinical utility and to examine the diagnostic and prognostic significance of the analysed biomarkers. The gene dosage pattern of the ERBB family and its downstream effectors PI3K and MYC showed particularly strong correlations with clinicopathological data. The ERBB PI3K/Akt pathway was upregulated in 31 (20%) of 156 cases. Activation of the ERBB PI3K/Akt pathway was positively correlated with a higher stage (p=0.001), higher grade (p=0.001), histological type II disease (p=0.0003) and metastases (p=0.02). The implemented hierarchical clustering revealed that cluster 2 was characterised by high copy numbers of the studied genes. Cluster 2 was associated with shorter overall survival (p=0.05). The platform was found to be a fast and simple method for direct analysis of the genes involved in uterine carcinogenesis, making it feasible for EC biology characterisation.

  10. Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

    Science.gov (United States)

    Obrero, Angeles; Die, Jose V; Román, Belén; Gómez, Pedro; Nadal, Salvador; González-Verdejo, Clara I

    2011-05-25

    The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

  11. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  12. Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

    DEFF Research Database (Denmark)

    Olsen, Morten Tange; Bérubé, Martine; Robbins, Jooke

    2012-01-01

    BACKGROUND:Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent...... critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting...... steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. RESULTS...

  13. Unfolding Green Defense

    DEFF Research Database (Denmark)

    Larsen, Kristian Knus

    2015-01-01

    to inform and support the further development of green solutions by unfolding how green technologies and green strategies have been developed and used to handle current security challenges. The report, initially, focuses on the security challenges that are being linked to green defense, namely fuel......In recent years, many states have developed and implemented green solutions for defense. Building on these initiatives NATO formulated the NATO Green Defence Framework in 2014. The framework provides a broad basis for cooperation within the Alliance on green solutions for defense. This report aims...... consumption in military operations, defense expenditure, energy security, and global climate change. The report then proceeds to introduce the NATO Green Defence Framework before exploring specific current uses of green technologies and green strategies for defense. The report concludes that a number...

  14. Counting mycobacteria in infected human cells and mouse tissue: a comparison between qPCR and CFU.

    Directory of Open Access Journals (Sweden)

    Sharad Pathak

    Full Text Available Due to the slow growth rate and pathogenicity of mycobacteria, enumeration by traditional reference methods like colony counting is notoriously time-consuming, inconvenient and biohazardous. Thus, novel methods that rapidly and reliably quantify mycobacteria are warranted in experimental models to facilitate basic research, development of vaccines and anti-mycobacterial drugs. In this study we have developed quantitative polymerase chain reaction (qPCR assays for simultaneous quantification of mycobacterial and host DNA in infected human macrophage cultures and in mouse tissues. The qPCR method cannot discriminate live from dead bacteria and found a 10- to 100-fold excess of mycobacterial genomes, relative to colony formation. However, good linear correlations were observed between viable colony counts and qPCR results from infected macrophage cultures (Pearson correlation coefficient [r] for M. tuberculosis = 0.82; M. a. avium = 0.95; M. a. paratuberculosis = 0.91. Regression models that predict colony counts from qPCR data in infected macrophages were validated empirically and showed a high degree of agreement with observed counts. Similar correlation results were also obtained in liver and spleen homogenates of M. a. avium infected mice, although the correlations were distinct for the early phase (< day 9 post-infection and later phase (≥ day 20 post-infection liver r = 0.94 and r = 0.91; spleen r = 0.91 and r = 0.87, respectively. Interestingly, in the mouse model the number of live bacteria as determined by colony counts constituted a much higher proportion of the total genomic qPCR count in the early phase (geometric mean ratio of 0.37 and 0.34 in spleen and liver, respectively, as compared to later phase of infection (geometric mean ratio of 0.01 in both spleen and liver. Overall, qPCR methods offer advantages in biosafety, time-saving, assay range and reproducibility compared to colony counting. Additionally, the duplex format allows

  15. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Andrzej J. Paszczynski; Ravindra Paidisetti; Andrew K. Johnson; Ronald L. Crawford; Frederick S. Colwell; Tonia Green; Mark Delwiche; Hope Lee; Deborah Newby; Eoin L. Brodie; Mark Conrad

    2011-11-01

    The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultraperformance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

  16. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Directory of Open Access Journals (Sweden)

    Susan D'Costa

    2016-01-01

    Full Text Available Clinical trials using recombinant adeno-associated virus (rAAV vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR is now the most common method to titer vector genomes (vg; however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  17. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

    OpenAIRE

    Michelle de Campos Soriani Azevedo; Natália Mortari Ramuno; Luciana Raquel Vincenzi Fachin; Mônica Tassa; Patrícia Sammarco Rosa; Andrea de Faria Fernandes Belone; Suzana Madeira Diório; Cleverson Teixeira Soares; Gustavo Pompermaier Garlet; Ana Paula Favaro Trombone

    2017-01-01

    Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique ...

  18. Real-time qPCR improves meningitis pathogen detection in invasive bacterial-vaccine preventable disease surveillance in Fiji

    OpenAIRE

    Dunne, Eileen M.; Mantanitobua, Silivia; Singh, Shalini P.; Reyburn, Rita; Tuivaga, Evelyn; Rafai, Eric; Tikoduadua, Lisi; Porter, Barbara; Satzke, Catherine; Strachan, Janet E.; Fox, Kimberly K.; Jenkins, Kylie M.; Jenney, Adam; Baro, Silo; Mulholland, E. Kim

    2016-01-01

    As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most co...

  19. Comparison of culture and qPCR for the detection of Pseudomonas aeruginosa in not chronically infected cystic fibrosis patients

    OpenAIRE

    Deschaght, Pieter; Schelstraete, Petra; Lopes dos Santos Santiago, Guido; Van Simaey, Leen; Haerynck, Filomeen; Van daele, Sabine; De Wachter, Elke; Malfroot, Anne; Lebecque, Patrick; Knoop, Christiane; Casimir, Georges; Boboli, Hedwige; Pierart, Fr?d?ric; Desager, Kristine; Vaneechoutte, Mario

    2010-01-01

    Abstract Background Pseudomonas aeruginosa is the major respiratory pathogen causing severe lung infections among CF patients, leading to high morbidity and mortality. Once infection is established, early antibiotic treatment is able to postpone the transition to chronic lung infection. In order to optimize the early detection, we compared the sensitivity of microbiological culture and quantitative PCR (qPCR) for the detection of P. aeruginosa in respiratory samples of not chronically infecte...

  20. Comparative evaluation of DNA extraction methods for amplification by qPCR of superficial vs intracellular DNA from Bacillus spores.

    Science.gov (United States)

    Brauge, Thomas; Faille, Christine; Inglebert, Gaëlle; Dubois, Thomas; Morieux, Paul; Slomianny, Christian; Midelet-Bourdin, Graziella

    2018-02-02

    This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification. Copyright © 2018

  1. Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR

    DEFF Research Database (Denmark)

    Dechesne, Arnaud; Musovic, Sanin; Palomo, Alejandro

    2016-01-01

    Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantit......RNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets....

  2. Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

    Science.gov (United States)

    Xi, Xiaoxia; Zhang, Jiachao; Kwok, Laiyu; Huo, Dongxue; Feng, Shuzhen; Zhang, Heping; Sun, Tiansong

    2015-12-01

    Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future.

  3. Comparison between conventional and qPCR methods for enumerating Campylobacter jejuni in a poultry processing plant.

    Science.gov (United States)

    Melero, Beatriz; Cocolin, Luca; Rantsiou, Kalliopi; Jaime, Isabel; Rovira, Jordi

    2011-10-01

    Campylobacter jejuni is worldwide recognized as a human foodborne pathogen. It is widely present in poultry meat and slaughterhouses, but little is known about its fate during the processing of poultry meat preparations. In stress conditions, this pathogen can enter into a viable but non-culturable state, where quantitative PCR (qPCR) becomes more convenient for its detection. In this study, two different pairs of primers, targeting the rpoB and the hipO genes, were compared for its detection and quantification by PCR. Two calibration curves were prepared: one for the meat samples and the other for the environmental samples. rpoB primers showed higher sensitivity with a quantification limit of 1 log cfu/g or ml. Microbial Assessment Scheme (MAS) was used to select the Critical Sampling Locations (CSLs) along the poultry processing line. Forty-six out of 48 samples were positive by qPCR after enrichment (t = 48 ) while only 6 samples were positive by ISO 10272-1:2006. Forty-three samples showed positive signal without enrichment (t = 0 h), however only 16 samples could be quantified. These results showed the high prevalence of C. jejuni in the poultry industry and the need for new, rapid and sensitive techniques, such as qPCR, for the detection and quantification of C. jejuni in meat and environmental samples. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods.

    Science.gov (United States)

    Olsen, Morten Tange; Bérubé, Martine; Robbins, Jooke; Palsbøll, Per J

    2012-09-06

    Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.

  5. Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

    Directory of Open Access Journals (Sweden)

    Olsen Morten

    2012-09-01

    Full Text Available Abstract Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause

  6. Green Power Partnership 100 Green Power Users

    Science.gov (United States)

    EPA's Green Power Partnership is a voluntary program designed to reduce the environmental impact of electricity generation by promoting renewable energy. Partners on this list use green power to meet 100 of their U.S. organization-wide electricity use.

  7. Green Power Markets

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership defines Green power is a subset of renewable energy and represents those renewable energy resources and technologies that provide the highest environmental benefit.

  8. Green Infrastructure Modeling Tools

    Science.gov (United States)

    Modeling tools support planning and design decisions on a range of scales from setting a green infrastructure target for an entire watershed to designing a green infrastructure practice for a particular site.

  9. Tribal Green Building Toolkit

    Science.gov (United States)

    This Tribal Green Building Toolkit (Toolkit) is designed to help tribal officials, community members, planners, developers, and architects develop and adopt building codes to support green building practices. Anyone can use this toolkit!

  10. Blue-Green Algae

    Science.gov (United States)

    ... people with hepatitis C or hepatitis B. HIV/AIDS. Research on the effects of blue-green algae in people with HIV/AIDS has been inconsistent. Some early research shows that taking 5 grams of blue-green ...

  11. Green Nail Syndrome

    Science.gov (United States)

    ... from blue-green to dark green to bluish-grey. Since the discoloration is underneath the nail, it ... infected by P. aeruginosa. Nails repeatedly immersed in water are susceptible. Housewives, dishwashers, cooks, and health care ...

  12. Green Bank Observatory (GBO)

    Data.gov (United States)

    Federal Laboratory Consortium — The largest fully steerable telescope in the world - the Robert C. Byrd Green Bank Telescope (GBT), began observations in Green Bank, West Virginia in 2000and is a...

  13. Decon Green (trademark)

    National Research Council Canada - National Science Library

    Wagner, George W; Procell, Lawrence R; Henderson, Vikki D; Sorrick, David C; Hess, Zoe A; Gehring, David G; Brickhouse, Mark D

    2004-01-01

    ...) is extremely environmentally friendly and leaves no toxic residues. Decon Green (trademark) retains the low-temperature decontamination ability of DS2, but it is noncorrosive to aluminum and steel. Decon Green (trademark...

  14. Green Power Communities

    Science.gov (United States)

    GPCs are towns, villages, cities, counties, or tribal governments in which the local government, businesses, and residents collectively use green power in amounts that meet or exceed EPA's Green Power Community purchase requirements.

  15. Green Power Partner List

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership is a voluntary program designed to reduce the environmental impact of electricity generation by promoting renewable energy. There are thousands of Green Power Partners, all listed on this page.

  16. The Green Man

    Science.gov (United States)

    Watson-Newlin, Karen

    2010-01-01

    The Jolly Green Giant. Robin Hood. The Bamberg Cathedral. Tales of King Arthur. Ecology. What do they have in common? What legends and ancient myths are shrouded in the tales of the Green Man? Most often perceived as an ancient Celtic symbol as the god of spring and summer, the Green Man disappears and returns year after year, century after…

  17. The green agenda

    CERN Document Server

    Calder, Alan

    2009-01-01

    This business guide to Green IT was written to introduce, to a business audience, the opposing groups and the key climate change concepts, to provide an overview of a Green IT strategy and to set out a straightforward, bottom line-orientated Green IT action plan.

  18. Green roof Malta

    OpenAIRE

    Gatt, Antoine

    2015-01-01

    In Malta, buildings cover one third of the Island, leaving greenery in the dirt track. Green roofs are one way to bring plants back to urban areas with loads of benefits. Antoine Gatt, who manages the LifeMedGreenRoof project at the University of Malta, tells us more. http://www.um.edu.mt/think/green-roof-malta/

  19. EPA's Green Roof Research

    Science.gov (United States)

    This is a presentation on the basics of green roof technology. The presentation highlights some of the recent ORD research projects on green roofs and provices insight for the end user as to the benefits for green roof technology. It provides links to currently available EPA re...

  20. Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters

    Data.gov (United States)

    U.S. Environmental Protection Agency — Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are...

  1. Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead in Biofilm Samples

    Directory of Open Access Journals (Sweden)

    Michael J. Taylor

    2014-01-01

    Full Text Available Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that

  2. Differential identification of Sporothrix spp. and Leishmania spp. by conventional PCR and qPCR in multiplex format.

    Science.gov (United States)

    Rodríguez-Brito, Sabrina; Camacho, Emma; Mendoza, Mireya; Niño-Vega, Gustavo A

    2015-01-01

    Sporotrichosis and cutaneous leishmaniasis are skin infections with similar clinical manifestations but different treatment methods. The present study aimed to evaluate qPCR and conventional PCR for differential detection of the etiological agents of both infections in multiplex format. Assays were designed using two sets of reported primers: SS1/SS2, designed on the 18S ribosomal RNA gene from Sporothrix spp., and JW11/JW12, designed on the kinetoplast DNA (kDNA) minicircles of Leishmania spp. qPCR detected 200 fg of DNA per reaction for both Sporothrix and Leishmania. Melting curve analysis revealed two distinctive Tm peaks for Sporothrix spp. (85.5°C), and Leishmania spp. (82.6°C). A detection limit of 20 pg was determined for the diagnosis of both with conventional PCR. No other clinically important organisms were detected by either PCR or qPCR. However, a Blast analysis on GenBank databases, using as query the sequence of the PCR fragment obtained with primers SS1/SS2, showed 100% identity to environmental fungi of the Ophiostomales order. Lower percentages of identity (≤80%), with mismatches at primers' sequence regions were obtained for other environmental or clinically important fungi. Proper handling of clinical samples is required to avoid false negatives due to contamination with environmental fungi of the Ophiostomales order. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Directory of Open Access Journals (Sweden)

    Andrea J Adams

    Full Text Available Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM to Macherey-Nagel DNA FFPE (MN, test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90% when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections, current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from

  4. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    Science.gov (United States)

    Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

  5. A novel, Q-PCR based approach to measuring endogenous retroviral clearance by capture protein A chromatography.

    Science.gov (United States)

    Zhang, Min; Lute, Scott; Norling, Lenore; Hong, Connie; Safta, Aurelia; O'Connor, Deborah; Bernstein, Lisa J; Wang, Hua; Blank, Greg; Brorson, Kurt; Chen, Qi

    2009-04-01

    Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

  6. The CIN4 chromosomal instability qPCR classifier defines tumor aneuploidy and stratifies outcome in grade 2 breast cancer

    DEFF Research Database (Denmark)

    Szász, Attila Marcell; Li, Qiyuan; Eklund, Aron Charles

    2013-01-01

    Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer. Methods: AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We......Purpose: Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene...

  7. Comparison among the Quantification of Bacterial Pathogens by qPCR, dPCR, and Cultural Methods

    Directory of Open Access Journals (Sweden)

    Matteo Ricchi

    2017-06-01

    Full Text Available The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR, has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad and the Quant Studio 3D (Applied Biosystems. The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was <0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative

  8. Q-PCR based bioburden assessment of drinking water throughout treatment and delivery to the International Space Station

    Science.gov (United States)

    Newcombe, David; Stuecker, Tara; La Duc, Myron; Venkateswaran, Kasthuri

    2005-01-01

    Previous studies indicated evidence of opportunistic pathogens samples obtained during missions to the International Space Station (ISS). This study utilized TaqMan quantitative PCR to determine specific gene abundance in potable and non-potable ISS waters. Probe and primer sets specific to the small subunit rRNA genes were used to elucidate overall bacterial rRNA gene numbers. while those specific for Burkholderia cepacia and Stenotrophomonas maltophilia were optimized and used to probe for the presence of these two opportunistic pathogens. This research builds upon previous microbial diversity studies of ISS water and demonstrates the utility of Q-PCR tool to examine water quality.

  9. Green energy in Europe: selling green energy with green certificates

    International Nuclear Information System (INIS)

    Ouillet, L.

    2002-01-01

    Sales of green power products are booming in Europe: 50,000 customers in the United Kingdom, 775,000 in the Netherlands and 300,000 in Germany. Laws of physics are however formal: the way in which electricity flows within the grid does not allow suppliers to assure customers that they are directly receiving electricity produced exclusively from renewable energy sources. What are marketers selling their customers then? Laetitia Ouillet, Greenprices, takes a closer look and focuses on the potential of selling green energy in the forms of renewable energy certificates. (Author)

  10. Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

    Science.gov (United States)

    Gholami, Khadijeh; Loh, Su Yi; Salleh, Naguib; Lam, Sau Kuen; Hoe, See Ziau

    2017-01-01

    Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

  11. Single-cell qPCR facilitates the optimization of hematopoietic differentiation in hPSCs/OP9 coculture system.

    Science.gov (United States)

    Chen, Haide; Jiang, Mengmeng; Xiao, Lei; Huang, He

    2018-03-15

    Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.

  12. Unraveling the Wheat Stem Rust Infection Process on Barley Genotypes Through Relative qPCR and Fluorescence Microscopy.

    Science.gov (United States)

    Zurn, J D; Dugyala, S; Borowicz, P; Brueggeman, R; Acevedo, M

    2015-05-01

    The infection process of wheat stem rust (Puccinia graminis f. sp. tritici) on barley (Hordeum vulgare) is often observed as a mesothetic infection type at the seedling stages, and cultivars containing the same major resistance genes often show variation in the level of resistance provided against the same pathogen race or isolate. Thus, robust phenotyping data based on quantification of fungal DNA can improve the ability to elucidate host-pathogen interaction, especially at early time points of infection when disease symptoms are not yet evident. Quantitative real-time polymerase chain reaction (qPCR) was used to determine the amount of fungal DNA relative to host DNA in infected tissue, providing new insights about fungal development and host resistance during the infection process in this pathosystem. The stem rust susceptible 'Steptoe', resistant cultivars containing only Rpg1 ('Beacon', 'Morex', and 'Chevron'), and the resistant line Q21861 containing Rpg1 and the rpg4/Rpg5 complex were evaluated using the traditional 0-to-4 rating scale, fluorescence microscopy, and qPCR. Statistical differences (Pwheat stem rust infection process, indicating potential host genotype contributions related to basal defense during the wheat stem rust infection process.

  13. Seasonal Dynamics of Microcystis spp. and Their Toxigenicity as Assessed by qPCR in a Temperate Reservoir

    Directory of Open Access Journals (Sweden)

    Agostinho Antunes

    2011-09-01

    Full Text Available Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes to non-toxic (without mcy genes genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4–6 weeks before and 8–10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems.

  14. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  15. Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; Botteldoorn, Nadine; Yde, Marc; Mahillon, Jacques; Roosens, Nancy H

    2013-05-01

    A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

  16. Green electricity buyer's guide

    International Nuclear Information System (INIS)

    Kelly, B.; Klein, S.; Olivastri, B.

    2002-06-01

    The electricity produced in whole or in large part from renewable energy sources like wind, small hydro electricity and solar energy, is generally referred to as green electricity. The authors designed this buyer's guide to assist customers in their understanding of green electricity, as the customers can now choose their electricity supplier. The considerations and steps involved in the purchasse of green electricity are identified, and advice is provided on ways to maximize the benefits from the purchase of green electricity. In Alberta and Ontario, customers have access to a competitive electricity market. The emphasis when developing this guide was placed firmly on the large buyers, as they can have enormous positive influence on the new market for green electricity. The first chapter of the document provides general information on green electricity. In chapter two, the authors explore the opportunity for environmental leadership. Chapter three reviews the basics of green electricity, which provides the link to chapter four dealing with the creation of a policy. Purchasing green electricity is dealt with in Chapter five, and maximizing the benefits of green electricity are examined in Chapter Six. 24 refs., 3 tabs

  17. Greening America's Communities

    Science.gov (United States)

    Technical assistance program to help cities and towns develop an implementable vision of distinctive, environmentally friendly neighborhoods using green infrastructure and other sustainable design strategies.

  18. The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance.

    Science.gov (United States)

    Gaby, John Christian; Buckley, Daniel H

    2017-10-01

    The measurement of functional gene abundance in diverse microbial communities often employs quantitative PCR (qPCR) with highly degenerate oligonucleotide primers. While degenerate PCR primers have been demonstrated to cause template-specific bias in PCR applications, the effect of such bias on qPCR has been less well explored. We used a set of diverse, full-length nifH gene standards to test the performance of several universal nifH primer sets in qPCR. We found significant template-specific bias in all but the PolF/PolR primer set. Template-specific bias caused more than 1000-fold mis-estimation of nifH gene copy number for three of the primer sets and one primer set resulted in more than 10,000-fold mis-estimation. Furthermore, such template-specific bias will cause qPCR estimates to vary in response to beta-diversity, thereby causing mis-estimation of changes in gene copy number. A reduction in bias was achieved by increasing the primer concentration. We conclude that degenerate primers should be evaluated across a range of templates, annealing temperatures, and primer concentrations to evaluate the potential for template-specific bias prior to their use in qPCR.

  19. QUANTITATIVE REAL-TIME PCR (Q-PCR FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

    Directory of Open Access Journals (Sweden)

    Yvana Maria Maia de Albuquerque

    2014-04-01

    Full Text Available Objective: To assess quantitative real-time polymerase chain reaction (q-PCR for the sputum smear diagnosis of pulmonary tuberculosis (PTB in patients living with HIV/AIDS with a clinical suspicion of PTB. Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard. Results: Of the 140 sputum samples, 47 (33.6% were positive with the gold standard. q-PCR was positive in 42 (30% of the 140 patients. Only one (0.71% did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93% of the 42 q-PCR positive cases, the CT (threshold cycle was equal to or less than 37. Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

  20. A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

    Directory of Open Access Journals (Sweden)

    Mateusz G Adamski

    Full Text Available Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1 the achievement of absolute quantification and (2 a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

  1. Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding.

    Science.gov (United States)

    Poulle, Marie-Lazarine; Forin-Wiart, Marie-Amélie; Josse-Dupuis, Émilie; Villena, Isabelle; Aubert, Dominique

    2016-01-01

    Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces. © M.-L. Poulle et al., published by EDP Sciences, 2016.

  2. Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia

    Czech Academy of Sciences Publication Activity Database

    Peková, Soňa; Marková, J.; Pajer, Petr; Dvořák, Michal; Cetkovský, P.; Schwarz, J.

    2005-01-01

    Roč. 9, č. 1 (2005), s. 23-34 ISSN 1084-8592 Institutional research plan: CEZ:AV0Z50520514 Keywords : minimal residual disease * chronic lymphocytic leukaemia * IgV(H) rearrangement Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.562, year: 2003

  3. Direct Comparison of the Histidine-rich Protein-2 Enzyme-linked Immunosorbent Assay (HRP-2 ELISA) and Malaria SYBR Green I Fluorescence (MSF) Drug Sensitivity Tests in Plasmodium falciparum Reference Clones and Fresh ex vivo Field Isolates from Cambodia

    Science.gov (United States)

    2013-07-12

    mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted...strains with varying drug susceptibility profiles: W2 and K1 (susceptible to mefloquine , and resistant to chloroquine), D6 (susceptible to chloroquine, and...resistant to mefloquine ), and 3D7 (susceptible to chloroquine, but less susceptible to mefloquine than W2). From the IC50 profiles of all parasite

  4. An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

    Science.gov (United States)

    Kawase, Jun; Etoh, Yoshiki; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2016-05-20

    Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

  5. Customers’ Intention to Use Green Products: the Impact of Green Brand Dimensions and Green Perceived Value

    Directory of Open Access Journals (Sweden)

    Doszhanov Aibek

    2015-01-01

    Full Text Available This study aimed to identify the relationships between green brand dimension (green brand awareness, green brand image, and green brand trust, green perceived value and customer’s intention to use green products. Data was collected through structured survey questionnaire from 384 customers of three hypermarkets in Kuala-Lumpur. Data was analyzed based on multiple regression analysis. The results indicate that there are significant relationships between green brand awareness, green brand trust, green perceived value, and customer’s intention to use green products. However, green brand image was not found to have significant relationship with customer’s intention to use green products. The discussion presented suggestions for marketers and researchers interested in green branding.

  6. Green Marketing amp Consumerism

    Directory of Open Access Journals (Sweden)

    Mohammad Jalalkamali

    2015-08-01

    Full Text Available Green marketing is a progressive issue that most of its dimensions are unknown or intangible for consumers. In this review some of its aspects are surveyed considering its impact on consumers as a fundamental segment in economics. Also consumer behavior is defined and analyzed through its awareness of green marketing issue which causes purchasing decision.

  7. Green Building Research Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Sailor, David Jean [Portland State Univ., Portland, OR (United States)

    2013-12-29

    This project provided support to the Green Building Research Laboratory at Portland State University (PSU) so it could work with researchers and industry to solve technical problems for the benefit of the green building industry. It also helped to facilitate the development of PSU’s undergraduate and graduate-level training in building science across the curriculum.

  8. The Greening Dutchman

    DEFF Research Database (Denmark)

    Arnold, Marlen Gabriele; Hockerts, Kai

    2011-01-01

    of ‘green flagging’ as a groundbreaking corporate sustainability innovation strategy. This paper describes how Philips uses this approach in its Green Flagship Program (GFP). Philips' GFP is particularly interesting since it sets specific targets across all its business units, thus driving the integration...... and limitations of these findings for theory and research on sustainability innovation strategies....

  9. Introduction: Experimental Green Strategies

    DEFF Research Database (Denmark)

    Peters, Terri

    2011-01-01

    Defining new ways in which archietcts are responding to the challenge of creating sustainable architecture , Experimental Green Strategies present a state of the art in applied ecological architectural research.......Defining new ways in which archietcts are responding to the challenge of creating sustainable architecture , Experimental Green Strategies present a state of the art in applied ecological architectural research....

  10. Green product innovation strategy

    NARCIS (Netherlands)

    Driessen, P.H.

    2005-01-01

    Over the last decades, companies have started to incorporate green issues in product innovation strategies. This dissertation studies green product innovation strategy, its antecedents and its outcomes. A three-stage approach is followed. In the first stage, the topic is explored and a preliminary

  11. Coordinate green growth

    NARCIS (Netherlands)

    Alkemade, F.; Hekkert, M.P.

    2010-01-01

    Green economic growth needs a shared sense of direction if it is to lead to a more sustainable future under climate change. Studies on green innovation and societal transformation show that uncoordinated initiatives are unlikely to be an effective way “to get the ball rolling and to ‘learn

  12. Green by Default

    DEFF Research Database (Denmark)

    Sunstein, Cass R.; Reisch, Lucia

    2013-01-01

    The article offers information on the two sources of energy including green energy and gray energy. It discusses several facts which includes lower levels of greenhouse gases and conventional pollutants, relationship between economic incentives and underlying preferences and potential effects of ...... of green default rules.....

  13. 10 Paths to Green

    Science.gov (United States)

    Kennedy, Mike

    2008-01-01

    Some institutions may feel comfortable with a few baby steps into the green world, while others may be ready to commit totally to environmental consciousness. Here, the author discusses 10 areas in which educators and administrators can beef up their green portfolio. These areas are in: alternative fuel, bikes/walking, water, education tools,…

  14. The Green Revolution.

    Science.gov (United States)

    Huke, Robert E.

    1985-01-01

    Modern agriculture's green revolution refers to a complex package that includes improved seeds and a wide range of efficient management practices. The genetic history of and technological developments that led to the green revolution are described, and its impact discussed. (RM)

  15. Green for rarity

    International Nuclear Information System (INIS)

    Raal, F.A.; Robinson, D.N.

    1980-01-01

    Green diamonds once recovered from Witwatersrand gold/uranium deposits, are now a thing of the past with the modernisation of extraction metallurgy methods. The green colouration has been shown to be due to radiation from uranium present in the ore

  16. Greening the Future

    Science.gov (United States)

    Williamson, Norma Velia

    2011-01-01

    Because educators vicariously touch the future through their students, the author believes that they sometimes have the uncanny ability to see the future. One common future forecast is the phenomenal growth of green jobs in the emerging green economy, leading to the creation of the "Reach of the Sun" Solar Energy Academy at La Mirada…

  17. Green Cleaning Label Power

    Science.gov (United States)

    Balek, Bill

    2012-01-01

    Green cleaning plays a significant and supportive role in helping education institutions meet their sustainability goals. However, identifying cleaning products, supplies and equipment that truly are environmentally preferable can be daunting. The marketplace is inundated with products and services purporting to be "green" or environmentally…

  18. Green Buildings and Health.

    Science.gov (United States)

    Allen, Joseph G; MacNaughton, Piers; Laurent, Jose Guillermo Cedeno; Flanigan, Skye S; Eitland, Erika Sita; Spengler, John D

    2015-09-01

    Green building design is becoming broadly adopted, with one green building standard reporting over 3.5 billion square feet certified to date. By definition, green buildings focus on minimizing impacts to the environment through reductions in energy usage, water usage, and minimizing environmental disturbances from the building site. Also by definition, but perhaps less widely recognized, green buildings aim to improve human health through design of healthy indoor environments. The benefits related to reduced energy and water consumption are well-documented, but the potential human health benefits of green buildings are only recently being investigated. The objective of our review was to examine the state of evidence on green building design as it specifically relates to indoor environmental quality and human health. Overall, the initial scientific evidence indicates better indoor environmental quality in green buildings versus non-green buildings, with direct benefits to human health for occupants of those buildings. A limitation of much of the research to date is the reliance on indirect, lagging and subjective measures of health. To address this, we propose a framework for identifying direct, objective and leading "Health Performance Indicators" for use in future studies of buildings and health.

  19. Green technology in Taiwan

    International Nuclear Information System (INIS)

    Runderkamp, D.

    2010-01-01

    Taiwan is striving for leadership in the field of green Technologies. Solar cells and Light Emitting Diodes (LED) are the best examples of successful Taiwanese green technologies. Electronic vehicles and smart grids are giving new impetus to the Taiwanese export of high-quality technology. [nl

  20. A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species.

    Directory of Open Access Journals (Sweden)

    Holger Linck

    Full Text Available Rubus stunt is an economically important disease in the production of raspberries, blackberries, and loganberries. A fast, sensitive, and reliable diagnosis of phytoplasmas, the causal agent of the disease, is of prime importance to stop its spread by vegetative propagation and by insect vectors. Therefore, multiplex qPCR assays using TaqMan probes with different kinds of fluorophores in one reaction were developed, allowing the detection of phytoplasmas in general as well as a more specific detection of phytoplasmas belonging to group 16SrV and host DNA (either plant or insect. This assay now provides a practical tool for the screening of motherplants and monitoring the presence and distribution of phytoplasmas in Rubus plants of different geographic origins, cultivars, and cultivation systems, as well as in putative insect vectors like leafhoppers.

  1. Early detection of sugar beet pathogen Ramularia beticola in leaf and air samples using qPCR

    DEFF Research Database (Denmark)

    Wieczorek, Thies Marten; Jørgensen, Lise Nistrup; Hansen, Anne Lisbet

    2014-01-01

    A quantitative PCR method (qPCR) was developed for the detection and quantification of Ramularia beticola causing Ramularia leaf spot in sugar beet. R. beticola specific primers were designed based on the internal transcribed spacer region 2 (ITS2). The assay was applied on DNA extracted from...... spores trapped on tape from Burkard spore traps placed in an artificially inoculated sugar beet field trial and in two sugar beet fields with natural infections. R. beticola DNA was detected at variable amounts in the air samples 14 to 16 days prior to first visible symptoms. R. beticola DNA was detected....... beticola in plant material and in air samples prior to the appearance of visible symptoms. An early detection has potential as a tool, which can be part of a warning system predicting the onset of the disease in the sugar beet crop and helping to optimise fungicide application....

  2. The utility of optical detection system (qPCR) and bioinformatics methods in reference gene expression analysis

    Science.gov (United States)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Real-time quantitative polymerase chain reaction is consider as the most reliable method for gene expression studies. However, the expression of target gene could be misinterpreted due to improper normalization. Therefore, the crucial step for analysing of qPCR data is selection of suitable reference genes, which should be validated experimentally. In order to choice the gene with stable expression in the designed experiment, we performed reference gene expression analysis. In this study genes described in the literature and novel genes predicted as control genes, based on the in silico analysis of transcriptome data were used. Analysis with geNorm and NormFinder algorithms allow to create the ranking of candidate genes and indicate the best reference for flower morphogenesis study. According to the results, genes CACS and CYCL were characterised the most stable expression, but the least suitable genes were TUA and EF.

  3. Quantification of B16 Melanoma Cells in Lungs Using Triplex Q-PCR - A New Approach to Evaluate Melanoma Cell Metastasis and Tumor Control

    DEFF Research Database (Denmark)

    Sorensen, Maria R; Pedersen, Sara R; Lindkvist, Annika

    2014-01-01

    of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found...... the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection......, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFNγ, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective...

  4. Green growth in fisheries

    DEFF Research Database (Denmark)

    Nielsen, Max; Ravensbeck, Lars; Nielsen, Rasmus

    2014-01-01

    harming the environment. Fishery is an environment-dependent sector and it has been argued that there is no potential for green growth in the sector owing to global overexploitation, leaving no scope for production growth. The purpose of this paper is to explain what green growth is and to develop...... a conceptual framework. Furthermore, the aim is to show that a large green growth potential actually exists in fisheries and to show how this potential can be achieved. The potential green growth appears as value-added instead of production growth. The potential can be achieved by reducing overcapacity......, investing in the rebuilding of fish stocks and a coordinated regulation of marine activities that interact with fisheries. Incentive-based regulation of fisheries that counterbalances services of the ecosystems is an important instrument to achieve green growth....

  5. Green heterogeneous wireless networks

    CERN Document Server

    Ismail, Muhammad; Nee, Hans-Peter; Qaraqe, Khalid A; Serpedin, Erchin

    2016-01-01

    This book focuses on the emerging research topic "green (energy efficient) wireless networks" which has drawn huge attention recently from both academia and industry. This topic is highly motivated due to important environmental, financial, and quality-of-experience (QoE) considerations. Specifically, the high energy consumption of the wireless networks manifests in approximately 2% of all CO2 emissions worldwide. This book presents the authors’ visions and solutions for deployment of energy efficient (green) heterogeneous wireless communication networks. The book consists of three major parts. The first part provides an introduction to the "green networks" concept, the second part targets the green multi-homing resource allocation problem, and the third chapter presents a novel deployment of device-to-device (D2D) communications and its successful integration in Heterogeneous Networks (HetNets). The book is novel in that it specifically targets green networking in a heterogeneous wireless medium, which re...

  6. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

    Science.gov (United States)

    Stokdyk, Joel P.; Firnstahl, Aaron; Spencer, Susan K.; Burch, Tucker R; Borchardt, Mark A.

    2016-01-01

    The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L−1 assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.

  7. Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR.

    Science.gov (United States)

    Stokdyk, Joel P; Firnstahl, Aaron D; Spencer, Susan K; Burch, Tucker R; Borchardt, Mark A

    2016-06-01

    The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L(-1) assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation. Published by Elsevier Ltd.

  8. Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

    Science.gov (United States)

    Pu, Fei; Yang, Bingye; Ke, Caihuan

    2015-07-01

    Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 ( ACT-2), elongation factor 1 alpha ( EF-1α), elongation factor 1 beta ( EF-1β), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), ubiquitin ( UBQ), β-tubulin ( β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene ( Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

  9. Seawater detection and biological assessments regarding transmission of the oyster parasite Mikrocytos mackini using qPCR.

    Science.gov (United States)

    Polinski, Mark P; Meyer, Gary R; Lowe, Geoffrey J; Abbott, Cathryn L

    2017-10-18

    Mikrocytos mackini is an intracellular parasite of oysters and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Although M. mackini has been investigated for decades, its natural mode of transmission, mechanism for host entry, and environmental stability are largely unknown. We explored these biological characteristics of M. mackini using a recently described quantitative PCR (qPCR) assay. We detected M. mackini in the flow-through tank water of experimentally infected oysters and during disease remission in host tissues following 6 wk of elevated water temperature. Waterborne exposure of oysters to M. mackini further confirmed the potential for extracellular seawater transmission of this parasite and also identified host gill to have the highest early and continued prevalence for M. mackini DNA compared to stomach, mantle, labial palps, or adductor muscle samples. However, infections following waterborne challenge were slow to develop despite a substantial exposure (>106 M. mackini l-1 for 24 h), and further investigation demonstrated that M. mackini occurrence and infectivity severely declined following extracellular seawater incubation of more than 24 h. This study demonstrates a potential for using qPCR to monitor M. mackini in wild or farmed oyster populations during periods of disease remission or from environmental seawater samples. This work also suggests that gill tissues may provide a primary site for waterborne entry and possibly shedding of M. mackini in oysters. Further, although extracellular seawater transmission of M. mackini was possible, poor environmental stability and infection efficiency likely restricts the geographic transmission of M. mackini between oysters in natural environs and may help to explain localized areas of infection.

  10. Building the green way.

    Science.gov (United States)

    Lockwood, Charles

    2006-06-01

    Just five or six years ago, the term "green building" evoked visions of barefoot, tie-dyed, granola-munching denizens. There's been a large shift in perception. Of course, green buildings are still known for conserving natural resources by, for example, minimizing on-site grading, using alternative materials, and recycling construction waste. But people now see the financial advantages as well. Well-designed green buildings yield lower utility costs, greater employee productivity, less absenteeism, and stronger attraction and retention of workers than standard buildings do. Green materials, mechanical systems, and furnishings have become more widely available and considerably less expensive than they used to be-often cheaper than their standard counterparts. So building green is no longer a pricey experiment; just about any company can do it on a standard budget by following the ten rules outlined by the author. Reliable building-rating systems like the U.S. Green Building Council's rigorous Leadership in Energy and Environmental Design (LEED) program have done much to underscore the benefits of green construction. LEED evaluates buildings and awards points in several areas, such as water efficiency and indoor environmental quality. Other rating programs include the UK's BREEAM (Building Research Establishment's Environmental Assessment Method) and Australia's Green Star. Green construction is not simply getting more respect; it is rapidly becoming a necessity as corporations push it fully into the mainstream over the next five to ten years. In fact, the author says, the owners of standard buildings face massive obsolescence. To avoid this problem, they should carry out green renovations. Corporations no longer have an excuse for eschewing environmental and economic sustainability. They have at their disposal tools proven to lower overhead costs, improve productivity, and strengthen the bottom line.

  11. Validation of a multiplex qPCR assay for the identification and quantification of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis: In vitro and subgingival plaque samples.

    Science.gov (United States)

    Marin, M J; Ambrosio, N; Herrera, D; Sanz, M; Figuero, E

    2018-04-01

    To validate a multiplex qPCR (m-qPCR) assay for the simultaneous identification and quantification of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in subgingival samples. In vitro samples: DNA combinations of A. actinomycetemcomitans and P. gingivalis in similar or different concentrations were prepared. qPCR and m-qPCR were performed using the same primers and hydrolysis probes specific for 16SrRNA genes. Results were analyzed using intra-class (ICCs) and Lin's correlation coefficients (r) based on quantification cycle (Cq) values. Subgingival plaque samples: a cross-sectional study analyzing subgingival plaque samples harvested from periodontally-healthy and chronic periodontitis patients. Samples were processed by either qPCR or m-qPCR targeting both bacteria. Sensitivity, specificity, predictive values and Lińs correlation coefficients (r) were calculated using CFU/mL as primary outcome. In vitro samples: m-qPCR yielded a good reproducibility (coefficients of variation around 1% and ICCs > 0.99) for both bacterial species. m-qPCR achieved detection limits and specificity similar to qPCR. An excellent concordance (r = 0.99) was observed between m-qPCR and qPCR for A. actinomycetemcomitans and P. gingivalis without statistical significant differences between both methods Subgingival plaque samples: a high sensitivity (above 80%) and specificity (100%) was obtained with the m-qPCR for both bacteria. The m-qPCR yielded a good concordance in Cq values, showing a good level of agreement between qPCR and m-qPCR. The tested m-qPCR method was successful in the simultaneous quantification of A. actinomycetemcomitans and P. gingivalis, with a high degree of sensitivity and specificity on subgingival plaque samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

    Science.gov (United States)

    Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

    2012-01-01

    We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions. PMID:22701644

  13. Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

    Directory of Open Access Journals (Sweden)

    Uffe Vest Schneider

    Full Text Available We introduce quantitative polymerase chain reaction (qPCR primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

  14. Collection Development "Green Business": The Green Capitalist

    Science.gov (United States)

    Eagan, Robert

    2009-01-01

    The "greening" of corporate behemoths like Wal-Mart, DuPont, and Toyota has received much media attention in recent years. But consider small businesses: according to the U.S. Small Business Administration, of the estimated 27 million firms in the United States, 99.7 percent have fewer than 500 employees, 97.5 percent have fewer than 20, and more…

  15. Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF

    DEFF Research Database (Denmark)

    Tratwal, Josefine; Follin, Bjarke; Ekblond, Annette

    2014-01-01

    on reference genes (RGs) for the normalization of qPCR data. RESULTS: BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation...... gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization...

  16. Harmonic oscillator Green's function

    International Nuclear Information System (INIS)

    Macek, J.H.; Ovchinnikov, S.Yu.; Khrebtukov, D.B.

    2000-01-01

    The Green's function for the harmonic oscillator in three dimensions plays an important role in the theory of atomic collisions. One representation of low-energy ion-atom collisions involves harmonic oscillator potentials. A closed-form expression for the harmonic oscillator Green's function, needed to exploit this representation, is derived. This expression is similar to the expression for the Coulomb Green's function obtained by Hostler and Pratt. Calculations of electron distributions for a model system of ion-atom collisions are reported to illustrate the theory.

  17. About green political parties

    Directory of Open Access Journals (Sweden)

    Orlović Slobodan P.

    2015-01-01

    Full Text Available In this work the author refers to some legal and political questions in connection with green political parties. Those questions cover: the ideology of green political parties, their number and influence, both in general and in Serbia. The first part of work is generally speaking about political parties - their definition, ideology, role and action. Main thesis in this work is that green political parties, by their appearance, were something new on the political scene. But quickly, because of objective and subjective reasons, they were changing original ideas and were beginning to resemble to all other political parties. In this way, they lost their vanguard and political alternativeness.

  18. Sustainable green urban planning: the Green Credit Tool

    NARCIS (Netherlands)

    Cilliers, E.J.; Diemont, E.; Stobbelaar, D.J.; Timmermans, W.

    2010-01-01

    Purpose – The Green Credit Tool is evaluated as a method to quantify the value of green-spaces and to determine how these green-space-values can be replaced or compensated for within urban spatial planning projects. Design/methodology/approach – Amersfoort Local Municipality created the Green Credit

  19. Green Sturgeon Acoustic Monitoring

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This database is used to hold tracking data for green sturgeon tagged in Central California. The data collection began in late 2002 and is ongoing.

  20. Introduction: Green Building Handbook

    CSIR Research Space (South Africa)

    Van Wyk, Llewellyn V

    2010-08-01

    Full Text Available By recognising the specific environmental challenges facing South Africa, mindful of the government‘s commitment to reducing South Africa‘s Greenhouse gas emissions, and acknowledging the need to build social cohesion, the Green Building Handbook...

  1. Green Building Standards

    Science.gov (United States)

    Many organizations have developed model codes or rating systems that communities may use to develop green building programs or revise building ordinances. Some of the major options are listed on this page.

  2. Green Turtle Trophic Ecology

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — SWFSC is currently conducting a study of green sea turtle (Chelonia mydas) trophic ecology in the eastern Pacific. Tissue samples and stable carbon and stable...

  3. Green Turtle Critical Habitat

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — These data represent the critical habitat for green turtle as designated by Federal Register Vol. 63, No. 46701, September 2, 1998, Rules and Regulations.

  4. Compliance for Green IT

    CERN Document Server

    Calder, Alan

    2009-01-01

    The growing range of Green IT regulations are challenging more and more organisations to take specific steps to ensure they are in compliance with sometimes complex regulations, ranging from cap & trade requirements through to regulations concerning IT equipment disposal.

  5. Expanding the Green Revolution.

    Science.gov (United States)

    Mellor, John W.; Riely, Frank Z.

    1989-01-01

    Described are some of the successes of the Green Revolution in third-world nations. Discussed are research priorities; misconceptions; and improvements in management skills, training and education, infrastructure, and international trade. (CW)

  6. The Green Revolution Game.

    Science.gov (United States)

    Corbridge, Stuart

    1985-01-01

    The Green Revolution game helps college students learn about agrarian change in which people use science to transform nature. The rational and basic objectives of the game are discussed, and the game's strengths and weaknesses are examined. (RM)

  7. Green certificates causing inconvenience?

    International Nuclear Information System (INIS)

    Torgersen, Lasse

    2002-01-01

    From early 2002, producers of green energy in selected countries have been able to benefit from generous financial support in the Netherlands. Thus, there has been increased sale of green certificates from Norway and Sweden. But the condition that physical energy delivery should accompany the certificates has caused a marked rise in the price of energy in transit through Germany to the Netherlands. This article discusses the green certificate concept and the experience gained from the Netherlands. One conclusion is that if large-scale trade with green certificates is introduced in Europe without the condition of accompanying energy delivery, then producers of hydro-electric power in Norway and Sweden may be the losers

  8. Manufacturing Green Consensus

    DEFF Research Database (Denmark)

    Gulsrud, Natalie Marie; Ooi, Can Seng

    2014-01-01

    In an increasingly global economy, being green, or having an environmentally sustainbale place brand, provides a competitive advantage. Singapore, long known as the ``garden city´´ has been a leader in green city imaging since the founding of the equatorial city-state, contributing, in large part...... to the city’s profile as the economic giant of Southeast Asia. Using a political ecology lens, the paper aims to uncover the contested socio-economic narratives of green city imaging by examining the evolution of the garden city branding scheme since Singapore’s independence in 1959. Results show...... that entrepreneurial governance such as green city branding has important and uneven political consequences for the social and economic fabric of our cities....

  9. Green Supplier Selection Criteria

    DEFF Research Database (Denmark)

    Nielsen, Izabela Ewa; Banaeian, Narges; Golinska, Paulina

    2014-01-01

    to determine prevalent general and environmental supplier selection criteria and develop a framework which can help decision makers to determine and prioritize suitable green supplier selection criteria (general and environmental). In this research we considered several parameters (evaluation objectives......) to establish suitable criteria for GSS such as their production type, requirements, policy and objectives instead of applying common criteria. At first a comprehensive and deep review on prevalent and green supplier selection literatures performed. Then several evaluation objectives defined to assess the green......Green supplier selection (GSS) criteria arise from an organization inclination to respond to any existing trends in environmental issues related to business management and processes, so GSS is integrating environmental thinking into conventional supplier selection. This research is designed...

  10. ADVANTAGES OF GREEN TECHNOLOGY

    OpenAIRE

    Ghanshyam Das Soni

    2017-01-01

    Technology is application of knowledge to practical requirements. Green technologies encompass various aspects of technology which help us reduce the human impact on the environment and create ways of sustainable development. Social equitability, economic feasibility and sustainability are the key parameters for green technologies. Today the environment is racing towards the tipping point at which we would have done permanent irreversible damage to the planet earth. Our current actions are pu...

  11. Towards a green economy

    CSIR Research Space (South Africa)

    Trotter, D

    2012-10-01

    Full Text Available a number of explicit and aligned green economic strategies, policies, and programmes; ? UNEP1 identifies, South Africa, Germany and China as having forward thinking policy showing a conscious effort to drive decoupling- with some evidence... review of unintended behavioural responses, counter incentives, and legal conflicts or obstacles in the context of a Green Economic transition, followed by appropriate reform; ? CSIR 2012 Slide 15 Enabling Funding and Investment Frameworks ? A review...

  12. Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods

    Science.gov (United States)

    The U.S.EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 Recreational Water Quality Criteria (RWQC). The CCE quantification unit stems from the calibration model used to ...

  13. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Møller, Michael Boe

    2011-01-01

    study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical...

  14. Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

    Science.gov (United States)

    Agrimonti, Caterina; Bottari, Benedetta; Sardaro, Maria Luisa Savo; Marmiroli, Nelson

    2017-09-08

    Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

  15. Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements

    Science.gov (United States)

    The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...

  16. Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments - poster

    Science.gov (United States)

    The U.S. Environmental Protection Agency (EPA) will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this or ...

  17. Human-associated fecal qPCR measurements and predicted risk of gastrointestinal illness in recreational waters contaminated with raw sewage

    Science.gov (United States)

    We used quantitative microbial risk assessment (QMRA) to estimate the risk of gastrointestinal (GI) illness associated with swimming in recreational waters containing different concentrations of human-associated fecal qPCR markers from raw sewage– HF183 and HumM2. The volume/volu...

  18. Empirical evaluation of humpback whale telomere length estimates : Quality control and factors causing variability in the singleplex and multiplex qPCR methods

    NARCIS (Netherlands)

    Olsen, Morten Tange; Berube, Martine; Robbins, Jooke; Palsboll, Per J.

    2012-01-01

    Background: Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent

  19. The guide to greening cities

    National Research Council Canada - National Science Library

    Johnston, Sadhu Aufochs

    2013-01-01

    ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Introduction: The New Urban Imperative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CHAPTER 1. Greening from the Inside: Stories...

  20. The sustainability of green funds

    NARCIS (Netherlands)

    Scholtens, B.

    This paper analyses the performance of the Dutch "Green Funds Scheme". This scheme is a policy instrument to advance green projects. The scheme relies on tax compensation for private investors who save or invest in green institutions below market returns. The green institutions select and monitor

  1. The Influence of Proactive Green Innovation and Reactive Green Innovation on Green Product Development Performance: The Mediation Role of Green Creativity

    Directory of Open Access Journals (Sweden)

    Yu-Shan Chen

    2016-09-01

    Full Text Available This study fills the research gap in the exploration of the relationships between both proactive and reactive green innovations and green product development performance, and examines the mediating effect of green creativity. Structural equation modeling (SEM is utilized to test the hypotheses. From the sample of 146 valid respondents, the results show that proactive green innovation positively affects green creativity and green product development performance, and green creativity positively affects green product development performance. In addition, our findings also indicate that the relationship between proactive green innovation and green product development performance is partially mediated by green creativity. Accordingly, green creativity plays a critical role for companies to achieve a great green product development performance. However, reactive green innovation does not significantly influence green creativity and green product development performance. Companies should develop proactive green innovation rather than reactive green innovation in order to enhance their green creativity and increase their product development performance.

  2. Development of a dry-reagent-based qPCR to facilitate the diagnosis of Mycobacterium ulcerans infection in endemic countries.

    Science.gov (United States)

    Babonneau, Jérémie; Bernard, Christian; Marion, Estelle; Chauty, Annick; Kempf, Marie; Robert, Raymond; Marsollier, Laurent

    2015-04-01

    Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection. We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix. Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.

  3. Quantifying mRNA and microRNA with qPCR in cervical carcinogenesis: a validation of reference genes to ensure accurate data.

    Directory of Open Access Journals (Sweden)

    Maria da Conceição Gomes Leitão

    Full Text Available A number of recent studies have catalogued global gene expression patterns in a panel of normal, tumoral cervical tissues so that potential biomarkers can be identified. The qPCR has been one of the most widely used technologies for detecting these potential biomarkers. However, few studies have investigated a correct strategy for the normalization of data in qPCR assays for cervical tissues. The aim of this study was to validate reference genes in cervical tissues to ensure accurate quantification of mRNA and miRNA levels in cervical carcinogenesis. For this purpose, some issues for obtaining reliable qPCR data were evaluated such as the following: geNorm analysis with a set of samples which meet all of the cervical tissue conditions (Normal + CIN1 + CIN2 + CIN3 + Cancer; the use of individual Ct values versus pooled Ct values; and the use of a single (or multiple reference genes to quantify mRNA and miRNA expression levels. Two different data sets were put on the geNorm to assess the expression stability of the candidate reference genes: the first dataset comprised the quantities of the individual Ct values; and the second dataset comprised the quantities of the pooled Ct values. Moreover, in this study, all the candidate reference genes were analyzed as a single "normalizer". The normalization strategies were assessed by measuring p16INK4a and miR-203 transcripts in qPCR assays. We found that the use of pooled Ct values, can lead to a misinterpretation of the results, which suggests that the maintenance of inter-individual variability is a key factor in ensuring the reliability of the qPCR data. In addition, it should be stressed that a proper validation of the suitability of the reference genes is required for each experimental setting, since the indiscriminate use of a reference gene can also lead to discrepant results.

  4. Development of a dry-reagent-based qPCR to facilitate the diagnosis of Mycobacterium ulcerans infection in endemic countries.

    Directory of Open Access Journals (Sweden)

    Jérémie Babonneau

    2015-04-01

    Full Text Available Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i highly resistant to heat; (ii of similar sensitivity and efficiency to the fresh mix and (iii easier to use than the fresh mix.Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.

  5. Evaluation of Real-Time Quantitative Polymerase Chain Reaction (qPCR) to Determine Escherichia coli Concentrations at Two Lake Erie Beaches

    Science.gov (United States)

    Kephart, Christopher M.; Bushon, Rebecca N.

    2009-01-01

    During the recreational seasons of 2006 and 2007, the quantitative polymerase chain reaction (qPCR) method was used to determine Escherichia coli (E. coli) concentrations in samples from two Lake Erie beaches. Results from the qPCR method were compared to those obtained by traditional culturing on modified mTEC agar. Regression analysis showed strong, statistically significant correlations between results from the two methods for both years. Correlation coefficients at Edgewater and Villa Angela Beaches were 0.626 and 0.789 for 2006 and 0.667 and 0.829 for 2007, respectively. Linear regression analyses were done to determine how well E. coli concentrations could have been predicted from qPCR results. These hypothetical predictions were compared to the current practice of determining recreational water quality from E. coli concentrations determined for samples collected on the previous day. The qPCR method resulted in a greater percentage of correct predictions of water-quality exceedances than the current method for both beaches and both years. However, because regression equations differed somewhat between both sites and both years, the study did not result in any single relation reliable enough to use for actual real-time prediction of water-quality exceedances for either beach; therefore, a posterior analysis of data was done. Additional years of data may be needed to develop such a relation. Results from this study support the continued development and testing of a qPCR method for providing rapid and accurate estimates of E. coli concentrations for monitoring recreational water quality.

  6. The Influence of Environmental Friendliness on Green Trust: The Mediation Effects of Green Satisfaction and Green Perceived Quality

    Directory of Open Access Journals (Sweden)

    Yu-Shan Chen

    2015-07-01

    Full Text Available As global green trends became more prevalent, green marketing also developed into an important issue. Although prior literature explored the main factors affecting green trust, it was inconclusive as to how environmental friendliness could affect the green trust in green marketing. This study aims to focus on the positive influence of environmental friendliness on green trust, and explore the mediation effects of green satisfaction and green perceived quality. This study undertakes an empirical study by means of questionnaire survey. The respondents are consumers who have experience purchasing green products. This study applies structural equation modeling (SEM to test the hypotheses. The findings of this study indicate that (1 environmental friendliness has a significant positive impact on green satisfaction, green perceived quality, and green trust; (2 both green satisfaction and green perceived quality positively affect green trust; and (3 green satisfaction and green perceived quality partially mediate the positive relationship between environmental friendliness and green trust.

  7. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA. Published by Elsevier Ireland Ltd.

  8. Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages.

    Science.gov (United States)

    Li, Meng-Yao; Wang, Feng; Jiang, Qian; Wang, Guan-Long; Tian, Chang; Xiong, Ai-Sheng

    2016-01-01

    A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

  9. Validation and comparison of reference genes for qPCR normalization of celery (Apium graveolens at different development stages

    Directory of Open Access Journals (Sweden)

    Meng-Yao eLi

    2016-03-01

    Full Text Available A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

  10. A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

    Science.gov (United States)

    Gallaher, Sean D.; Berk, Arnold J.

    2013-01-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity. PMID:23624118

  11. Green technology meets ecotoxicology

    Directory of Open Access Journals (Sweden)

    Kristina Radošević

    2016-01-01

    Full Text Available By applying concept and principles of green chemistry into different technological processes, green technologies are developed. The environmental and economic benefits of “green” approach is achieved through several directions, such as the use of renewable raw materials, creation of economic efficiency, the use of alternative reaction conditions, as well as the application of non-conventional solvents. From the point view of green chemistry, alternative solvents, in order to be a “green“ substitution to hazardous organic solvents, should be: non-volatile, non-flammable, stabile, synthesized by an environmentally friendly procedure, nontoxic and biodegradable. The toxic impact of all newly synthesized chemicals, such as alternative solvents, could be determined by methods and techniques of ecotoxicology. Ecotoxicology, an interdisciplinary scientific field, can serve as a way of monitoring the greenness of the processes. In vivo and in vitro experiments are used to study the effects of chemicals on different levels of organizations, from molecules to communities and ecosystem. The usage of in vitro methods is encouraged by a scientific community and regulatory agencies as an alternative to in vivo studies in order to reduce the number of laboratory animals used in the toxicological studies. Therefore, in this paper we gave a brief overview on the usage of animal cell cultures within the field of green chemistry and technology.

  12. GREEN BANKING IN ROMANIA

    Directory of Open Access Journals (Sweden)

    Dumitrascu Mihaela

    2014-07-01

    Full Text Available The paper investigates the green banking in Romania, a new approach of conducting the banking business through considering the corporate social responsibility and environmental aspects. Nowadays, it is difficult to face the globalization and competition in order to asssure the implementation of the green banking practices. The aim of the present study is to identify corporations that have sustainability concerns. To achieve this objective, we set some hypothesis and after this we showed that the corporations are more likely to be included in the list of top banks in the world. Our study is relevant for future research in this area, because of the importance of such aspects in corporations nowadays.The conclusions of our study is that green banking practices in Romania is in an incipient stage

  13. Green buildings pay

    DEFF Research Database (Denmark)

    Naboni, Emanuele; Edwards, Brian

    The new edition of ‘Green Buildings Pay’ authored by Brian Edwards and Emanuele Naboni explores the business and professional benefits which derive from architectural design driven by sustainability. With a new sub-title ‘Green Buildings Pay: design, productivity and ecology’ the book argues...... or environmental thinking and this finds expression in new approaches to the design of building facades, roofs, atria. Another is that new software simulation tools have changed energy assumptions and hence building forms. In a fast evolving arena, the book shows how architects are reshaping their practices....... Branding via LEED and BREEAM has taken green ideas to China and other emerging economies. The globalization of sustainability and of architectural practice is an important strand of the new edition....

  14. Certified: green power

    International Nuclear Information System (INIS)

    Rhodes, S.; Brown, L.

    1999-01-01

    Deregulation of the energy industry in the USA may be a force favouring the environment but for the consumer it is something of a nightmare since there are so many options with respect to both price, service and environmental awareness. However, there is now a marked tendency for companies wishing to be seen as 'green' to favour environmentally aware suppliers. Indeed, some suppliers holding formal qualifications in 'greenness' believe they are justified in charging a premium for their energy. The question is asked 'what is green?' and the authors discuss the answers at some length: the hydro industry fares well in such a discussion. The authors (from Scientific Certification Systems) believe that certification provides a rational explanation of prices and why charging a premium may be justifiable.(UK)

  15. Green buildings pay

    DEFF Research Database (Denmark)

    Naboni, Emanuele; Edwards, Brian

    The new edition of ‘Green Buildings Pay’ authored by Brian Edwards and Emanuele Naboni explores the business and professional benefits which derive from architectural design driven by sustainability. With a new sub-title ‘Green Buildings Pay: design, productivity and ecology’ the book argues...... that environmental design has altered how we design, construct and manage buildings. The book has relevance to those who not only design and engineer buildings but to those who commission architecture and those who occupy the products of this process. Hence, the user is a key consideration. The book examines via....... Branding via LEED and BREEAM has taken green ideas to China and other emerging economies. The globalization of sustainability and of architectural practice is an important strand of the new edition....

  16. Green Logistics Management

    Science.gov (United States)

    Chang, Yoon S.; Oh, Chang H.

    Nowadays, environmental management becomes a critical business consideration for companies to survive from many regulations and tough business requirements. Most of world-leading companies are now aware that environment friendly technology and management are critical to the sustainable growth of the company. The environment market has seen continuous growth marking 532B in 2000, and 590B in 2004. This growth rate is expected to grow to 700B in 2010. It is not hard to see the environment-friendly efforts in almost all aspects of business operations. Such trends can be easily found in logistics area. Green logistics aims to make environmental friendly decisions throughout a product lifecycle. Therefore for the success of green logistics, it is critical to have real time tracking capability on the product throughout the product lifecycle and smart solution service architecture. In this chapter, we introduce an RFID based green logistics solution and service.

  17. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent Strains of Ralstonia solanacearum.

    Directory of Open Access Journals (Sweden)

    Michael J Stulberg

    Full Text Available Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2 cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species strains (and a non-plant associated R. mannitolilytica, and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  18. Green Views of Marx

    Directory of Open Access Journals (Sweden)

    Sheryl D. Breen

    2014-01-01

    Full Text Available Is Marxist theory relevant or conducive to environmental political thought? This article considers responses to this question and the debate regarding possibilities of a red/green coalition through examination of four green theorists who represent positions of theoretical and historical significance: first, the argument that an enlightened reinterpretation of Marx reveals him to be an inherently ecological thinker; second, the belief that Marxist thought holds significant value for environmental theory but that crucial weaknesses demand essential revision; third, the rejection of Marx and his political theory as antagonistic to environmental protection; and, finally, social ecology’s use of dialectical naturalism to transcend Marx’s failings.

  19. Green in software engineering

    CERN Document Server

    Calero Munoz, Coral

    2015-01-01

    This is the first book that presents a comprehensive overview of sustainability aspects in software engineering. Its format follows the structure of the SWEBOK and covers the key areas involved in the incorporation of green aspects in software engineering, encompassing topics from requirement elicitation to quality assurance and maintenance, while also considering professional practices and economic aspects. The book consists of thirteen chapters, which are structured in five parts. First the "Introduction" gives an overview of the primary general concepts related to Green IT, discussing wha

  20. Green Chemistry Pedagogy

    Science.gov (United States)

    Kolopajlo, Larry

    2017-02-01

    This chapter attempts to show how the practice of chemistry teaching and learning is enriched by the incorporation of green chemistry (GC) into lectures and labs. To support this viewpoint, evidence from a wide range of published papers serve as a cogent argument that GC attracts and engages both science and nonscience students, enhances chemistry content knowledge, and improves the image of the field, while preparing the world for a sustainable future. Published pedagogy associated with green and sustainable chemistry is critically reviewed and discussed.

  1. Green Maritime Logistics

    DEFF Research Database (Denmark)

    Psaraftis, Harilaos N.

    2016-01-01

    By green maritime logistics we mean achieving an acceptable environmental performance of the maritime transport logistical supply chain while at the same time respecting traditional economic criteria. In this paper the environmental focus is on maritime emissions. Achieving such goal may involve...... several trade-offs, and win-win solutions are typically sought. However, finding these solutions may be more difficult than may appear at first glance. The purpose of this paper is to provide a concise overview of the challenges of green maritime logistics and present some examples, both for greenhouse...

  2. Green Chemistry Metrics with Special Reference to Green Analytical Chemistry

    OpenAIRE

    Marek Tobiszewski; Mariusz Marć; Agnieszka Gałuszka; Jacek Namieśnik

    2015-01-01

    The concept of green chemistry is widely recognized in chemical laboratories. To properly measure an environmental impact of chemical processes, dedicated assessment tools are required. This paper summarizes the current state of knowledge in the field of development of green chemistry and green analytical chemistry metrics. The diverse methods used for evaluation of the greenness of organic synthesis, such as eco-footprint, E-Factor, EATOS, and Eco-Scale are described. Both the well-establis...

  3. Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms.

    Science.gov (United States)

    Plusquin, Michelle; DeGheselle, Olivier; Cuypers, Ann; Geerdens, Ellen; Van Roten, Andromeda; Artois, Tom; Smeets, Karen

    2012-03-01

    The flatworm species Schmidtea mediterranea and Macrostomum lignano have become new and innovative model organisms in stem cell, regeneration and tissue homeostasis research. Because of their unique stem cell system, (lab) technical advantages and their phylogenetic position within the Metazoa, they are also ideal candidate model organisms for toxicity assays. As stress and biomarker screenings are often performed at the transcriptional level, the aim of this study was to establish a set of reference genes for qPCR experiments for these two model organisms in different stress situations. We examined the transcriptional stability of nine potential reference genes (actb, tubb, ck2, cox4, cys, rpl13, gapdh, gm2ap, plscr1) to assess those that are most stable during altered stress conditions (exposure to carcinogenic metals and salinity stress). The gene expression stability was evaluated by means of geNorm and NormFinder algorithms. Sets of best reference genes in these analyses varied between different stress situations, although gm2ap and actb were stably transcribed during all tested combinations. In order to demonstrate the impact of bad normalisation, the stress-specific gene hsp90 was normalised to different sets of reference genes. In contrast to the normalisation according to GeNorm and NormFinder, normalisation of hsp90 in Macrostomum lignano during cadmium stress did not show a significant difference when normalised to only gapdh. On the other hand an increase of variability was noticed when normalised to all nine tested reference genes together. Testing appropriate reference genes is therefore strongly advisable in every new experimental condition.

  4. A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems.

    Science.gov (United States)

    Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y

    2018-03-01

    DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

  5. Green Infrastructure Checklists and Renderings

    Science.gov (United States)

    Materials and checklists for Denver, CO to review development project plans for green infrastructure components, best practices for inspecting and maintaining installed green infrastructure. Also includes renderings of streetscape projects.

  6. Green Building Tools for Tribes

    Science.gov (United States)

    Tribal green building tools and funding information to support tribal building code adoption, healthy building, siting, energy efficiency, renewable energy, water conservation, green building materials, recycling and adaptation and resilience.

  7. Benefits of Green Power Partnership

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership is a voluntary partnership program designed to reduce the environmental impact of electricity generation by promoting renewable energy. Learn about the benefits of becoming a Green Power Partner.

  8. Green Manufacturing Fundamentals and Applications

    CERN Document Server

    2013-01-01

    Green Manufacturing: Fundamentals and Applications introduces the basic definitions and issues surrounding green manufacturing at the process, machine and system (including supply chain) levels. It also shows, by way of several examples from different industry sectors, the potential for substantial improvement and the paths to achieve the improvement. Additionally, this book discusses regulatory and government motivations for green manufacturing and outlines the path for making manufacturing more green as well as making production more sustainable. This book also: • Discusses new engineering approaches for manufacturing and provides a path from traditional manufacturing to green manufacturing • Addresses regulatory and economic issues surrounding green manufacturing • Details new supply chains that need to be in place before going green • Includes state-of-the-art case studies in the areas of automotive, semiconductor and medical areas as well as in the supply chain and packaging areas Green Manufactu...

  9. The Green Paper Issue.

    Science.gov (United States)

    Bourke, Paul; And Others

    1988-01-01

    Sixteen articles discuss the implications of Australia's Green Paper, a public policy statement recommending substantial changes in the higher education system to improve access. The issues discussed include labor force development, educational equity, women's education, and the financing of higher education. (MSE)

  10. Concept of Green Chemistry

    Indian Academy of Sciences (India)

    Srimath

    decades much work has been going on in this direction. The term. Green Chemistry was coined in 1991 by Anastas. ... She is currently working on electrochemical and insecticidal/antifungal activities for ..... less substances. Inherently Safer Chemistry for Accident Prevention: Design chemicals and their forms (solid, liquid, ...

  11. Lean Green Machines

    Science.gov (United States)

    Villano, Matt

    2011-01-01

    Colleges and universities have been among the leaders nationwide in adopting green initiatives, partly due to their demographics, but also because they are facing their own budget pressures. Virtualization has become the poster child of many schools' efforts, because it provides significant bang for the buck. However, more and more higher…

  12. Green, Brown, and probability

    CERN Document Server

    Chung, Kai Lai

    1995-01-01

    This volume shows modern probabilistic methods in action: Brownian Motion Process as applied to the electrical phenomena investigated by Green et al., beginning with the Newton-Coulomb potential and ending with solutions by first and last exits of Brownian paths from conductors.

  13. Revisiting Lexington Green.

    Science.gov (United States)

    Scheurman, Geoffrey

    1998-01-01

    Identifies and discusses different ways in which teachers using constructivist and other approaches might teach a lesson on the Lexington Green incident of April 1775. In that incident British soldiers opened fire on colonial farmers, killing eight of them. Includes excerpts from eyewitness documents and other background material. (MJP)

  14. Developing Green Line Products

    DEFF Research Database (Denmark)

    Muñoz-Marin, Ana Maria; Lassen, Astrid Heidemann; Poulsen, Søren Bolvig

    2011-01-01

    This publication is based on the Master thesis “User-driven ecoinnovation process: Towards the implementation of the Green product line at JELD-WEN” written by Ana Maria Muñoz-Marin as her Graduation Project for the MSc. Global Innovation Management degree. The company-based experiment was carried...

  15. Being 'green' helps profitability?

    International Nuclear Information System (INIS)

    Austin, D.

    1999-01-01

    Pollution reduction beyond regulatory compliance is gaining momentum among firms, but managers ask if being 'green' helps profitability. Evidence suggests it doesn't hurt, but when we see environmentally attractive firms with sound financial performance, it cannot yet say which is cause and which is effect [it

  16. Picturing Urban Green Attachments

    DEFF Research Database (Denmark)

    Blok, Anders; Meilvang, Marie Leth

    2015-01-01

    In this article, we explore the cultural-political tensions and ambiguities of urban ecology, by way of following how activists move and translate between ‘familiar’ and ‘public’ engagements in the green city. Empirically, we locate our exploration in and around Nordhavnen (The North Harbor...

  17. Greening China Naturally

    Science.gov (United States)

    Shixiong Cao; Ge Sun; Zhiqiang Zhang; Liding Che; Qi Feng; et. al.

    2011-01-01

    China leads the world in afforestation, and is one of the few countries whose forested area is increasing. However, this massive “greening” effort has been less effective than expected; afforestation has sometimes produced unintended environmental, ecological, and socioeconomic consequences, and has failed to achieve the desired ecological benefits. Where afforestation...

  18. Between green and grey

    NARCIS (Netherlands)

    Jeanet Kullberg

    2016-01-01

    Original title: Tussen groen en grijs Taking cuttings is cool. Growing vegetables is all the rage. Green oases can now be found scattered throughout Dutch towns and cities: community gardens and roof gardens where residents can go to relax and enjoy themselves, improve the appearance of their

  19. Why Green Taxation

    DEFF Research Database (Denmark)

    Hjøllund, Lene; Svendsen, Gert Tinggaard

    2001-01-01

    According to economists solving environmental problems is simple. Politicians should simply impose a uniform tax on harmful emissions. However, the actual design of such green taxation shows that politicians do not follow their advice. CO2 taxation in OECD, for example, is highly differentiated...

  20. Green Software Products

    NARCIS (Netherlands)

    Jagroep, E.A.

    2017-01-01

    The rising energy consumption of the ICT industry has triggered a quest for more green, energy efficient ICT solutions. The role of software as the true consumer of power and its potential contribution to reach sustainability goals has increasingly been acknowledged. At the same time, it is shown to

  1. Green corridors basics

    DEFF Research Database (Denmark)

    Panagakos, George

    2016-01-01

    The purpose of this chapter is to introduce the concept of ‘green corridors’ as a means to develop integrated, efficient and environmentally friendly transportation of freight between major hubs and by relative long distances. The basis of this material is work conducted in the context of the EU ...

  2. News: Green Chemistry & Technology

    Science.gov (United States)

    A series of 21 articles focused on different features of green chemistry in a recent issue of Chemical Reviews. Topics extended over a wide range to include the design of sustainable synthetic processes to biocatalysis. A selection of perspectives follows as part of this colu

  3. Plants as green phones

    NARCIS (Netherlands)

    Soler, R.; Harvey, J.A.; Bezemer, T.M.; Stuefer, J.F.

    2008-01-01

    Plants can act as vertical communication channels or `green phones¿ linking soil-dwelling insects and insects in the aboveground ecosystem. When root-feeding insects attack a plant, the direct defense system of the shoot is activated, leading to an accumulation of phytotoxins in the leaves. The

  4. Concept of Green Chemistry

    Indian Academy of Sciences (India)

    Srimath

    supercritical at 374 oC and 218 Atm. Hence, this clean and cheap solvent is used as a green solvent for many synthetic reactions. The reactions in ionic liquids need no special apparatus and methodologies, and they can be recycled. Supercritical CO2 fluid is now becoming an important commercial and industrial solvent.

  5. Green Vehicle Guide

    Science.gov (United States)

    ... about MPG math Discover fuel-saving tips Promote green vehicles Your mileage may vary $ 0 0 , 0 0 0 , 0 0 0 , 0 0 0 GHG metric tons Check out the estimated savings of U.S. consumers who have already purchased new vehicles under the fuel economy & greenhouse gas standard! More about the standards » Check ...

  6. Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

    Science.gov (United States)

    Schoener, E R; Hunter, S; Howe, L

    2017-07-01

    Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

  7. A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

    Science.gov (United States)

    Pulverer, Walter; Hofner, Manuela; Preusser, Matthias; Dirnberger, Elisabeth; Hainfellner, Johannes A; Weinhaeusel, Andreas

    2014-01-01

    MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

  8. Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

    Science.gov (United States)

    León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

    2017-01-01

    Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

  9. GREEN PACKAGING, GREEN PRODUCT, GREEN ADVERTISING, PERSEPSI, DAN MINAT BELI KONSUMEN

    Directory of Open Access Journals (Sweden)

    Imam Santoso

    2016-12-01

    Full Text Available Environmental problems become one of the strategic issues in achieving global competitiveness. One of the issues is products that are made from environmental friendly materials or known as green product. Furthermore, in green products marketing, the company also uses green packaging and green advertising concept. This study aimed to analyze the effect of green packaging, green products, and green advertising on consumer perception and purchasing intention. The study was conducted in Ketawanggede Village, Lowokwaru Sub-district, Malang City. The sampling method used nonprobability accidential sampling techniques. The numbers of respondents were 113 consumers in study site. Data were collected by interview using questionnaires. The method of analysis used Generalized Structured Component Analysis (GSCA. The analysis showed that the green packaging, green products, and green advertising had positive significant influence on consumer perceptions. Meanwhile, green product and consumer perception had positive significant influence on purchasing interest, but the green packaging and green advertising has not found sufficient evidence in influencing purchasing intention.

  10. Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo

    Directory of Open Access Journals (Sweden)

    Micol Falabella

    2017-10-01

    Full Text Available Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-associated protein 9 (Cas9-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA probes combined with an internal reference probe (Drop-Off Assay provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations.

  11. Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events in Vivo

    Science.gov (United States)

    Falabella, Micol; Sun, Linqing; Barr, Justin; Pena, Andressa Z.; Kershaw, Erin E.; Gingras, Sebastien; Goncharova, Elena A.; Kaufman, Brett A.

    2017-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations. PMID:28860183

  12. Selection of reference genes for gene expression analysis by real-time qPCR in avian cells infected with infectious bronchitis virus.

    Science.gov (United States)

    Batra, Ambalika; Maier, Helena J; Fife, Mark S

    2017-04-01

    Infectious bronchitis virus (IBV) causes infectious bronchitis in poultry, a respiratory disease that is a source of major economic loss to the poultry industry. Detection and the study of the molecular pathogenesis of the virus often involve the use of real-time quantitative PCR assays (qPCR). To account for error within the experiments, the levels of target gene transcription are normalized to that of suitable reference genes. Despite publication of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines in 2009, single un-tested reference genes are often used for normalization of qPCR assays in avian research studies. Here, we use the geNorm algorithm to identify suitable reference genes in different avian cell types during infection with apathogenic and pathogenic strains of IBV. We discuss the importance of selecting an appropriate experimental sample subset for geNorm analysis, and show the effect that this selection can have on resultant reference gene selection. The effects of inappropriate normalization on the transcription pattern of a cellular signalling gene, AKT1, and the interferon-inducible, MX1, were studied. We identify the possibility of the misinterpretation of qPCR data when an inappropriate normalization strategy is employed. This is most notable when measuring the transcription of AKT1, where changes are minimal during infection.

  13. Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops.

    Science.gov (United States)

    Luo, Y; Gu, S; Felts, D; Puckett, R D; Morgan, D P; Michailides, T J

    2017-02-01

    To develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. This study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. For Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 5·60 to 6·94 during the 16 days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. This study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants. © 2016 The Society for Applied Microbiology.

  14. A theoretical introduction to "combinatory SYBRGreen qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants.

    Science.gov (United States)

    Van den Bulcke, Marc; Lievens, Antoon; Barbau-Piednoir, Elodie; MbongoloMbella, Guillaume; Roosens, Nancy; Sneyers, Myriam; Casi, Amaya Leunda

    2010-03-01

    The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.

  15. Green Schools on Ordinary Budgets

    Science.gov (United States)

    Hanson, Mark E.

    2010-01-01

    Some in the green building industry have spoken for some time now of green buildings not needing to cost more. Jason McLennan in his 2004 book "The Philosophy of Sustainable Design" discusses not falling into the "green is always more" syndrome. He goes on to explain the concept of tunneling through the cost barrier. A 2007…

  16. It's Not Easy Building Green.

    Science.gov (United States)

    Higgins, Joseph

    2003-01-01

    Discusses green buildings, facilities designed, constructed, and operated in an environmentally friendly and resource-efficient way. Discusses reasons for campuses to "go green," the "shades of green" or variations in environmental-friendliness, certification through the Leadership in Energy and Environmental Design (LEED) rating system, financial…

  17. Green Roofs for Stormwater Management

    Science.gov (United States)

    This project evaluated green roofs as a stormwater management tool. Results indicate that the green roofs are capable of removing 40% of the annual rainfall volume from a roof through retention and evapotranspiration. Rainfall not retained by green roofs is detained, effectively...

  18. Guide to Purchasing Green Power

    Science.gov (United States)

    The Guide for Purchasing Green Power is a comprehensive guide for current and potential buyers of green power with information about green power purchasing. The Guide is created cooperatively between the EPA, the U.S. Department of Energy, the World Resou

  19. Green economy and related concepts

    NARCIS (Netherlands)

    Loiseau, Eleonore; Saikku, Laura; Antikainen, Riina; Droste, Nils; Hansjürgens, Bernd; Pitkänen, Kati; Leskinen, Pekka; Kuikman, Peter; Thomsen, Marianne

    2016-01-01

    For the last ten years, the notion of a green economy has become increasingly attractive to policy makers. However, green economy covers a lot of diverse concepts and its links with sustainability are not always clear. In this article, we focus on definitions of green economy and related concepts

  20. Investing in a green future

    Science.gov (United States)

    Clapp, Christa

    2018-01-01

    The growing green bond market reflects the financial sector's awakening to climate risk. New research examining the US municipal bond market suggests a positive green bond premium in recent years, driven by differences in credit quality. As climate-risk disclosure becomes more widespread, investors may show willingness to pay green premiums.

  1. Green Consumerism : an Eco-Friendly Behaviour Form Through The Green Product Consumption and Green Marketing

    OpenAIRE

    Handayani, Wiwik

    2017-01-01

    This research is referred to analyse the influence of consumer attitude of green product towards purchase intention. The consumer attitude of green product is a psychological tendencies that is expressed by evaluating a certain entity with some advantage or disadvantage considerations. The problem of this research is the low of cunsumer awareness to consume green product, because the lack to comprehend the importance of green product usage for health and eco-friendly. The purpose of this rese...

  2. Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

    OpenAIRE

    Gosiewski, Tomasz; Flis, Agnieszka; Sroka, Agnieszka; K?dzierska, Anna; Pietrzyk, Agata; K?dzierska, Jolanta; Drwi?a, Rafa?; Bulanda, Ma?gorzata

    2014-01-01

    Background Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT? 3D blood culture system (bioM?rieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. Results Using qPCR, FISH, SF, and culture, mi...

  3. LEMming: A Linear Error Model to Normalize Parallel Quantitative Real-Time PCR (qPCR Data as an Alternative to Reference Gene Based Methods.

    Directory of Open Access Journals (Sweden)

    Ronny Feuer

    Full Text Available Gene expression analysis is an essential part of biological and medical investigations. Quantitative real-time PCR (qPCR is characterized with excellent sensitivity, dynamic range, reproducibility and is still regarded to be the gold standard for quantifying transcripts abundance. Parallelization of qPCR such as by microfluidic Taqman Fluidigm Biomark Platform enables evaluation of multiple transcripts in samples treated under various conditions. Despite advanced technologies, correct evaluation of the measurements remains challenging. Most widely used methods for evaluating or calculating gene expression data include geNorm and ΔΔCt, respectively. They rely on one or several stable reference genes (RGs for normalization, thus potentially causing biased results. We therefore applied multivariable regression with a tailored error model to overcome the necessity of stable RGs.We developed a RG independent data normalization approach based on a tailored linear error model for parallel qPCR data, called LEMming. It uses the assumption that the mean Ct values within samples of similarly treated groups are equal. Performance of LEMming was evaluated in three data sets with different stability patterns of RGs and compared to the results of geNorm normalization. Data set 1 showed that both methods gave similar results if stable RGs are available. Data set 2 included RGs which are stable according to geNorm criteria, but became differentially expressed in normalized data evaluated by a t-test. geNorm-normalized data showed an effect of a shifted mean per gene per condition whereas LEMming-normalized data did not. Comparing the decrease of standard deviation from raw data to geNorm and to LEMming, the latter was superior. In data set 3 according to geNorm calculated average expression stability and pairwise variation, stable RGs were available, but t-tests of raw data contradicted this. Normalization with RGs resulted in distorted data contradicting

  4. Live-dead discrimination analysis, qPCR assessment for opportunistic pathogens, and population analysis at ozone wastewater treatment plants.

    Science.gov (United States)

    Jäger, Thomas; Alexander, Johannes; Kirchen, Silke; Dötsch, Andreas; Wieland, Arne; Hiller, Christian; Schwartz, Thomas

    2018-01-01

    In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Conventional wastewater treatment plants are normally not equipped with advanced technologies for the elimination of bacteria. Molecular biology analyses were combined with live-dead discrimination analysis of wastewater population using Propidium monoazide (PMA) to study population shifts during ozonation (1 g ozone/g DOC) at a municipal wastewater treatment plant. Escherichia coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas a reduction was observed for E. coli and enterococci. Results comparing conventional cultivation experiments with PMA-qPCR underlined the presence of viable but not culturable cells (VBNC) and their regrowth potential after ozone treatment. Illumina HiSeq sequencing results with and without PMA treatment demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and also underlined the importance of PMA treatment for the evaluation of elimination and selection processes during ozonation at WWTPs. Amongst a number of 14 most abundant genera identified in the inflow samples, 9 genera were found to be reduced, whereas 4 genera increased in relative abundance and 1 genus almost remained constant. The strongest increase in relative abundance after ozonation was detected for Oscillatoria spp., Microcoleus spp. and Nitrospira spp. Beside this, a continuous release of Pseudomonas spp. (including P. aeruginosa) to the downstream receiving body was confirmed. Regrowth experiments demonstrated a high prevalence of P. aeruginosa as part of the surviving bacterial population. Summing

  5. Green roofs; Les toitures vegetalisees

    Energy Technology Data Exchange (ETDEWEB)

    Seghier, C.

    2006-03-15

    Impervious surface coverage keeps spreading in cities. Streets, sidewalks, parking lots and roofs are waterproof, meaning greater amounts of water to channel and treat and higher flood risks during heavy rainfalls. Green roofing can play a key part in addressing this alarming issue. There are three types of green roofs: extensive, semi-intensive and intensive. The extensive green roof technique uses a thin soil covering with a variety of species providing year-round plant coverage. The plants are not necessarily horticultural in which case routine maintenance is minimal. No watering is needed. Usually extensive green roofs create an ecosystem. The semi-intensive green roof technique uses a soil covering of average thickness and serves to create decorative roofing. Although maintenance is moderate, watering is essential. The intensive green roof technique produces a terrace roof garden. Another advantage of green roofs is they increase the life cycle of the sealing. Roof sealing protection may see the span of its life cycle, now at about fifteen years, doubled if the building has a green roof. planning professionals still know very little about green roofing solutions. Yet, green roofing provides unquestionable ecological qualities and thermal and acoustic performance that have proven to be environmentally friendly. Yet France lags behind northern European countries in green roofing. The Germans, Swiss, Austrians, Scandinavians and Dutch have been using the technique for more than twenty years. (A.L.B.)

  6. Green and lean management

    CERN Document Server

    Davim, J

    2017-01-01

    This book focusses on the challenges and changes organizational management faces in an era when the need to develop environmentally aware processes meets high levels of competition. It covers the synergetic effects, how re-use, recycling, waste reduction, and other sustainable production strategies can add value, low costs and time of production. Sustainable business behavior is not only an environmental perspective on management, but more and more contains an organizational perspective. Taking into account these issues, green and lean management appears as the way managers can drive their employees to continuously improve the management processes that add value to the organization and costumers. This book provides information on principles, strategies, models, and applications of green and lean management, and at the same time communicates the latest research activity relating to this scientific field world-wide.

  7. The green protest

    International Nuclear Information System (INIS)

    Brun, R.

    1978-01-01

    The increasing public interest in the 'green' and the 'coloured' made the politicians alert. The ecological movement and the public initiatives show their effects. What 'utopists', 'crazy people' think about large technical development gains public importance. The uneasiness about a social development which was directed only by increasing spending power and permanent expansion, begins to articulate itself. Values such as solidarity and community, considerateness for fellow men and environment which had been eliminated in the past, are regaining their meaning. From the point of view of this steadily increasing movement, the phenomenon of the 'green parties' is investigated. In this context, the persons involved give their opinions, as well as representatives of the established parties, thus giving this volume the character of a documentation on an important trend in today's events. (orig.) [de

  8. Towards green construction

    International Nuclear Information System (INIS)

    Bajracharya, Bijaya B.; Shrestha, Prasanna M.

    2000-01-01

    Sustainability is the key to any development works. In the operation phase, hydro power is the most sustainable form of energy. However construction activities for the same power station are usually far from being green. The popular myth is that construction activity converts green into grey. Despite this popular myth, construction of a hydro power project in Nepal has made the project area greener than earlier during the construction phase itself. Choice of construction technology, appropriate level of environmental impact assessment, monitoring of environmental parameters along side the construction progress followed by mitigation at the right time; launching community development programmes side by side, having environmental specification in contractual documents and self-reliance to fulfill environmental obligations by contractors itself are the key factors in the environmental management within the construction activities. The main conclusions in the paper is the need to change the way to think about the project constraints

  9. Green chemistry: development trajectory

    International Nuclear Information System (INIS)

    Moiseev, I I

    2013-01-01

    Examples of applications of green chemistry methods in heavy organic synthesis are analyzed. Compounds, which can be produced by the processing of the biomass, and the criteria for the selection of the most promising products are summarized. The current status of the ethanol production and processing is considered. The possibilities of the use of high fatty acid triglycerides, glycerol, succinic acid, and isoprene are briefly discussed. The bibliography includes 67 references

  10. Green dentistry: The future

    Directory of Open Access Journals (Sweden)

    Mensudar Rathakrishnan

    2017-01-01

    Full Text Available Dentistry is a profession dedicated to promote health and wellness of the people. Green dentistry is a relatively new emerging concept in dentistry. Most dental offices are privately-owned small businesses and have no financial advantage to invest in many environmental-friendly practices. For this reason, more research is needed to find cost-effective environmental alternatives in dentistry. The aim of this article is to expand awareness and practice of eco-friendly dentistry.

  11. Green chemistry: development trajectory

    Science.gov (United States)

    Moiseev, I. I.

    2013-07-01

    Examples of applications of green chemistry methods in heavy organic synthesis are analyzed. Compounds, which can be produced by the processing of the biomass, and the criteria for the selection of the most promising products are summarized. The current status of the ethanol production and processing is considered. The possibilities of the use of high fatty acid triglycerides, glycerol, succinic acid, and isoprene are briefly discussed. The bibliography includes 67 references.

  12. of Brilliant Green

    Directory of Open Access Journals (Sweden)

    Rana Seyrani

    2016-01-01

    Full Text Available Novel hydrogel nanocomposites, based on κ-carrageenan polysaccharide, were prepared by graft copolymerization of acrylamide (AAM and maleic anhydride (MAH as comonomers in the presence of multiwall carbon nanotubes (MWCNT, using methylene bisacrylamide (MBA and ammonium persulfate (APS,former as a crosslinking agent and the latter as an initiator. The hydrogel nanocomposites structure was characterized by FTIR spectroscopy, scanning electron microscopy (SEM and XRD patterns, and their thermal stability was investigated by TGA thermal analysis. The hydrogel nanocomposites were evaluated using gel content measurements and swelling rate in distilled water and in saline solutions. The carbon nanotube content was examined in relation to its effect on the properties of nanocomposites. The results showed that with increasing carbon nanotube content, the rate of water absorbency and equilibrium swelling in distilled water decreased whereas the water absorbency in the saline solutions increased. Water retention capacity was also studied and the results indicated that the inclusion of carbon nanotube increased water retention under heating condition. Furthermore, the experimental conditions of adsorption kinetics and dynamics for the removal of cationic dye, Brilliant Green (BG, were studied in the range of 6-8 for pH, 10-60 min for time (t, and 10-300 mg/L for initial concentration (C0 of the dye. The optimum conditions obtained for adsorption of Brilliant Green dye were pH 7, t= 50 min and C0= 10 mg/L. Also, the results indicated that more than 98% of the maximum adsorption capacity toward Brilliant Green dye was achieved within the initial 10 min. The experimental tests showed that the hydrogels could be used as fast–responsive and high capacity sorbents in Brilliant Green removal processes from industrial waste water.

  13. INL Green Building Strategy

    Energy Technology Data Exchange (ETDEWEB)

    Jennifer Dalton

    2005-05-01

    Green buildings, also known as sustainable buildings, resource efficient buildings, and high performance buildings, are structures that minimize the impact on the environment by using less energy and water, reducing solid waste and pollutants, and limiting the depletion of natural resources. As Idaho National Laboratory (INL) becomes the nation’s premier nuclear energy research laboratory, the physical infrastructure will be established to help accomplish the mission. This infrastructure, particularly the buildings, should incorporate green design features in order to be environmentally responsible and reflect an image of progressiveness and innovation to the public and prospective employees. With this in mind, the recommendations described in this strategy are intended to form the INL foundation for green building standards. The recommendations in this strategy are broken down into three levels: Baseline Minimum, Leadership in Energy and Environmental Design (LEED)Certification, and Innovative. Baseline Minimum features should be included in all new occupied buildings no matter what the purpose or size. These features do not require significant research, design, or capital costs and yet they can reduce Operation and Maintenance (O&M) costs and produce more environmentally friendly buildings. LEED Certification features are more aggressive than the Baseline Minimums in that they require documentation, studies, and/or additional funding. Combined with the Baseline Minimums, many of the features in this level will need to be implemented to achieve the goal of LEED certification. LEED Silver certification should be the minimum goal for all new buildings (including office buildings, laboratories, cafeterias, and visitor centers) greater than 25,000 square feet or a total cost of $10 million. Innovative features can also contribute to LEED certification, but are less mainstream than those listed in the previous two levels. These features are identified as areas where

  14. The Influence of Consumers Perception of Green Products on Green Purchase Intention

    OpenAIRE

    Wilson Kong; Amran Harun; Rini Suryati Sulong; Jaratin Lily

    2014-01-01

    Green consumerism has increasingly received attention since the increased level of consumer awareness towards green products. Therefore, the aim of this paper had been to examine the influence of consumer perception of green products on green purchase intention. In this study, perception of green products was conceptualized as a multidimensional variable comprised of green corporate perception, eco-label, green advertising, green packaging, and green product value. By using a survey, a total ...

  15. Green Chemistry Metrics with Special Reference to Green Analytical Chemistry.

    Science.gov (United States)

    Tobiszewski, Marek; Marć, Mariusz; Gałuszka, Agnieszka; Namieśnik, Jacek

    2015-06-12

    The concept of green chemistry is widely recognized in chemical laboratories. To properly measure an environmental impact of chemical processes, dedicated assessment tools are required. This paper summarizes the current state of knowledge in the field of development of green chemistry and green analytical chemistry metrics. The diverse methods used for evaluation of the greenness of organic synthesis, such as eco-footprint, E-Factor, EATOS, and Eco-Scale are described. Both the well-established and recently developed green analytical chemistry metrics, including NEMI labeling and analytical Eco-scale, are presented. Additionally, this paper focuses on the possibility of the use of multivariate statistics in evaluation of environmental impact of analytical procedures. All the above metrics are compared and discussed in terms of their advantages and disadvantages. The current needs and future perspectives in green chemistry metrics are also discussed.

  16. Green Chemistry Metrics with Special Reference to Green Analytical Chemistry

    Directory of Open Access Journals (Sweden)

    Marek Tobiszewski

    2015-06-01

    Full Text Available The concept of green chemistry is widely recognized in chemical laboratories. To properly measure an environmental impact of chemical processes, dedicated assessment tools are required. This paper summarizes the current state of knowledge in the field of development of green chemistry and green analytical chemistry metrics. The diverse methods used for evaluation of the greenness of organic synthesis, such as eco-footprint, E-Factor, EATOS, and Eco-Scale are described. Both the well-established and recently developed green analytical chemistry metrics, including NEMI labeling and analytical Eco-scale, are presented. Additionally, this paper focuses on the possibility of the use of multivariate statistics in evaluation of environmental impact of analytical procedures. All the above metrics are compared and discussed in terms of their advantages and disadvantages. The current needs and future perspectives in green chemistry metrics are also discussed.

  17. The Green Experiment: Cities, Green Stormwater Infrastructure, and Sustainability

    Directory of Open Access Journals (Sweden)

    Christopher M. Chini

    2017-01-01

    Full Text Available Green infrastructure is a unique combination of economic, social, and environmental goals and benefits that requires an adaptable framework for planning, implementing, and evaluating. In this study, we propose an experimental framework for policy, implementation, and subsequent evaluation of green stormwater infrastructure within the context of sociotechnical systems and urban experimentation. Sociotechnical systems describe the interaction of complex systems with quantitative and qualitative impacts. Urban experimentation—traditionally referencing climate change programs and their impacts—is a process of evaluating city programs as if in a laboratory setting with hypotheses and evaluated results. We combine these two concepts into a singular framework creating a policy feedback cycle (PFC for green infrastructure to evaluate municipal green infrastructure plans as an experimental process within the context of a sociotechnical system. After proposing and discussing the PFC, we utilize the tool to research and evaluate the green infrastructure programs of 27 municipalities across the United States. Results indicate that green infrastructure plans should incorporate community involvement and communication, evaluation based on project motivation, and an iterative process for knowledge production. We suggest knowledge brokers as a key resource in connecting the evaluation stage of the feedback cycle to the policy phase. We identify three important needs for green infrastructure experimentation: (i a fluid definition of green infrastructure in policy; (ii maintenance and evaluation components of a green infrastructure plan; and (iii communication of the plan to the community.

  18. Buying and selling green: deregulation and green power marketing

    International Nuclear Information System (INIS)

    Evans, Andrew

    2000-01-01

    This article discusses the increasing trend towards deregulation of electricity markets, and the driving forces for liberalisation in the EU and North America. The use of green tariffs offered by utilities to differentiate themselves from competitors and to gain and keep customers is reported, and the situation with regard to green energy within the deregulated electricity markets in Australia, the EU, Denmark, Finland, Germany, the Netherlands, Portugal, Sweden, the UK, Canada and the USA is outlined. Customers switching as a result of green tariffs, the growing role of renewables, and opportunities for the promotion of green tariffs are discussed. (UK)

  19. The Influence of Proactive Green Innovation and Reactive Green Innovation on Green Product Development Performance: The Mediation Role of Green Creativity

    OpenAIRE

    Yu-Shan Chen; Tai-Wei Chang; Chun-Yu Lin; Pi-Yu Lai; Kuan-Hung Wang

    2016-01-01

    This study fills the research gap in the exploration of the relationships between both proactive and reactive green innovations and green product development performance, and examines the mediating effect of green creativity. Structural equation modeling (SEM) is utilized to test the hypotheses. From the sample of 146 valid respondents, the results show that proactive green innovation positively affects green creativity and green product development performance, and green creativity positivel...

  20. Commercial green energy. Final report

    International Nuclear Information System (INIS)

    Kalweit, B.

    1998-11-01

    Firms offering a Green electricity product have discovered that residential customers are willing to pay extra for the assurance that their electricity is generated through the use of non-polluting or renewable resources. This research investigated the market potential for Green energy at the next level of the energy consuming chain, commercial establishments at which small and medium sized businesses interface with customers. Green energy is proving to be an attractive proposition to some consumers in the residential marketplace. Is there a possibility that Green energy can also be sold to commercial enterprises? This research project sought to answer this question and to investigate the factors that might lead small business people to opt for Green. Answers to these questions will help energy companies target the businesses most likely to accept Green power with the right product set and product features

  1. Design of a novel quantitative PCR (QPCR-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds gene

    Directory of Open Access Journals (Sweden)

    Coleman Michael P

    2007-10-01

    Full Text Available Abstract Background Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR. Results We have developed a rapid, robust and efficient genotyping method for Wlds using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wlds mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wlds mutation as well as animals expressing the Wlds transgene. Conclusion We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number. This technique will be of particular benefit in studies where Wlds mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wlds mutation.

  2. Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds) gene

    Science.gov (United States)

    Wishart, Thomas M; MacDonald, Stephen HF; Chen, Philip E; Shipston, Michael J; Coleman, Michael P; Gillingwater, Thomas H; Ribchester, Richard R

    2007-01-01

    Background Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR). Results We have developed a rapid, robust and efficient genotyping method for Wlds using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wlds mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wlds mutation as well as animals expressing the Wlds transgene. Conclusion We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number. This technique will be of particular benefit in studies where Wlds mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wlds mutation. PMID:17971231

  3. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Directory of Open Access Journals (Sweden)

    Cengiz Ikten

    Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  4. Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto.

    Science.gov (United States)

    Kulik, Tomasz; Ostrowska, Anna; Buśko, Maciej; Pasquali, Matias; Beyer, Marco; Stenglein, Sebastian; Załuski, Dariusz; Sawicki, Jakub; Treder, Kinga; Perkowski, Juliusz

    2015-10-01

    An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r(2)-values of >0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

    Science.gov (United States)

    Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

    2012-01-01

    During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

  6. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Science.gov (United States)

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  7. Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems.

    Directory of Open Access Journals (Sweden)

    Ana L M Lacerda

    Full Text Available Quantitative Polymerase Chain Reaction (qPCR is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L. genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes ('Santa Clara', susceptible, and its near-isogenic line 'LAM 157', resistant; subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV and its mock-inoculated control and in two distinct times after inoculation (early and late. Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper. To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.

  8. The trashing of Big Green

    International Nuclear Information System (INIS)

    Felten, E.

    1990-01-01

    The Big Green initiative on California's ballot lost by a margin of 2-to-1. Green measures lost in five other states, shocking ecology-minded groups. According to the postmortem by environmentalists, Big Green was a victim of poor timing and big spending by the opposition. Now its supporters plan to break up the bill and try to pass some provisions in the Legislature

  9. Green Tourism Marketing Model1

    OpenAIRE

    Hasan, Ali

    2015-01-01

    Green Tourism Marketing Model research as efforts to develop environmentally friendly tourism destination, the synergy of government, business and community participation become the driving force of tourism product development with highly competitive. In the long term, this research aims to provide the marketing concept of green tourism as economic development efforts and strengthen the environment (eco-growth) through the development of green tourism marketing models. The ...

  10. Green Consumption Behavior Antecedents

    DEFF Research Database (Denmark)

    Pagiaslis, Anastasios; Krystallis Krontalis, Athanasios

    2014-01-01

    The present study adds to the evolving literature on green consumer behavior by examining through statistically robust methods the effect and interrelationships of the key constructs of environmental concern, consumer environmental knowledge, beliefs about biofuels, and behavioral intention (i...... for the environment has a positive and direct impact on environmental knowledge, beliefs, and behavioral intention. Also, demographics determine levels of concern for the environment and environmental knowledge. All constructs associate positively with one another delineating that the interdependencies between them...... are important when accounting for environmental behavior. Future research should validate present results with the use of cross-cultural samples and investigate whether environmental concern increases due to social desirability response bias....

  11. Green Arctic Patrol Vessel

    Science.gov (United States)

    2011-08-01

    Complement Flexible space for hovercraft , airboats, USVs and RHIBs Naval Surface Warfare Center Carderock Division Green Arctic Patrol Vessel 1...through Sea State 3 (SS3). Flexible capability for a variety of organic craft, such as boat, hovercraft and/or Unmanned Underwater Vehicles (UUVs...2.0, Finland 6.0 2.7 2.8 6 1.8 Hovercraft Griffon/500TD 8.04 3.92 2.41 99 5 ~1.5 Hovercraft Griffon/2000TD 12.7 6.1 3.93 450 20

  12. Rapid Identification of Bacterial Antibiotic Resistance by qPCR in Infants with Gram-Negative Septicaemia: A Proof-of-Concept Study.

    Science.gov (United States)

    Lam, Hugh Simon; Chan, Kathy Y Y; Ip, Margaret; Leung, Kam Tong; Lo, Norman W S; Wong, Raymond P O; Li, Karen; Ng, Pak Cheung

    2017-01-01

    Neonatal sepsis remains an important cause of neonatal morbidity and mortality. Tools to rapidly predict antibiotic resistance in neonatal sepsis would be extremely valuable. To develop quantitative polymerase chain reaction (qPCR) primer/probe sets that can rapidly detect antibiotic resistance genes common to a neonatal unit, and to investigate the feasibility of direct detection of antibiotic resistance genes in whole blood of infants with Gram-negative septicaemia without first isolating the organism. Primer/probe sets were designed to detect genes that produce aminoglycoside-modifying enzymes or extended-spectrum β-lactamase. In phase 1, Gram-negative organisms isolated from neonatal clinical specimens within a 12-month period were analysed by qPCR to detect preselected genes. In phase 2, blood specimens of infants with Gram-negative septicaemia were subjected to qPCR analysis to detect antibiotic resistance genes for comparison against conventional antibiotic resistance profile results. Two primer/probe sets showed promising diagnostic utilities for the prediction of antibiotic resistance; the diagnostic utilities (sensitivity, specificity, positive predictive value and negative predictive value) were 90.9, 96.4, 92.6 and 95.5%, respectively, for AAC3-2 [aac(3')-IIa/aacC3/aacC2, aac(3')-IIc/aacC2] to detect gentamicin resistance, and 59.3, 99.3, 94.1 and 92.6%, respectively, for BLA-C1 (blaCTX-M-9, blaCTX-M-14, blaCTX-M-24, blaCTX-M-27) to detect cephalosporin resistance. Twenty-six infants were tested in phase 2, and both gentamicin and cephalosporin resistance patterns were predicted with 100% sensitivity and 100% specificity by AAC3-2 and BLA-C1, respectively. qPCR with appropriately designed primer/probe sets can predict antibiotic resistance directly from neonatal blood, and it can substantially reduce the turnaround time for antibiotic resistance results. © 2016 S. Karger AG, Basel.

  13. The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity

    DEFF Research Database (Denmark)

    Hermann-Bank, Marie Louise; Skovgaard, Kerstin; Stockmarr, Anders

    2013-01-01

    to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the intestinal microbiota, to be used with the high-throughput qPCR chip ‘Access Array 48.48′, AA48.48, (Fluidigm...... and diarrhoeic status. 454-sequencing confirmed the specificity of the primer sets. Diarrhoea was associated with a reduced number of members from the genus Streptococcus, and in particular S. alactolyticus. Conclusion The Gut Microbiotassay provides fast and affordable high-throughput quantification...

  14. The Geography of Green Power

    Energy Technology Data Exchange (ETDEWEB)

    OShaughnessy, Eric J [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Heeter, Jenny S [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Volpi, Christina M [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2017-10-25

    Green power refers to the voluntary purchase of renewable electricity by retail electricity customers. Green power is unlike compliance-based renewable energy procurement imposed by law or regulation. In 2016, over six million customers procured about 95 million megawatt-hours (MWh) of green power in the United States, which represents about 28% of all U.S. renewable energy sales, excluding large hydropower. In this fact sheet, we use available data to illustrate the geography of green power demand (in terms of number of customers) and supply (in terms of MWh of generation) by state.

  15. Toronto green roof construction standard

    International Nuclear Information System (INIS)

    Aster, D.

    2007-01-01

    Toronto City Council adopted a green roof strategy in February 2006. This paper reviewed the by-law governing the strategy as well as the work in progress to develop minimum standards for the design and construction of green roofs in Toronto. The strategy included a series of recommendations regarding the installation of green roofs on city buildings; a pilot grant program; using the development process to encourage green roofs; and, public education and promotion. It was noted that compared to Europe, the development of standards for green roofs in North America is in its early stages. As an emerging sustainable technology, there currently are no standards incorporated into Ontario's Building Code against which Toronto can measure the design and construction of green roofs. Therefore this paper included an analysis detailing how the recommended design requirements were able to support the City's green roof policy objectives and integrate the performance criteria for green roofs previously established and supported by Toronto City Council. The key policy objectives of the City's green roof strategy were to reduce the urban heat island effect; to address stormwater management implications in terms of quality and quantity; to improve the energy budgets of individual buildings; and, to improve air quality

  16. The guide to greening cities

    National Research Council Canada - National Science Library

    Johnston, Sadhu Aufochs

    2013-01-01

    ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 CHAPTER 3. Leading in the Community: Using City Assets, Policy, Partnerships, and Persuasion . . Case in Point: Returning to Green City Roots and Loving El...

  17. Product management in green markets

    Directory of Open Access Journals (Sweden)

    Čajka Zoran

    2005-01-01

    Full Text Available The present paper deals with the concept of green product management. To create a significantly greener economy, there will need to be a range of new and greener products and technologies. Today we are faced with a growth in more innovative "clean technology" solutions. Successful development of new green products requires high levels of communication and integration, good information, early consideration of green issues, support from top management, and benchmarking. The set of controllable tactical marketing tools (product, price, place and promotion that the company blends to produce the response it wants in the target green market, is the matter of the primary importance to the management.

  18. Ecological Transition and Green Investment

    International Nuclear Information System (INIS)

    Bureau, Dominique

    2016-01-01

    By introducing an exhaustible resource into an AK growth model, we can identify the determinants of the scale of green investment needs and its impact on capital accumulation dynamics. The role of green capital in the transition to a low-carbon economy depends, in particular, on the relative magnitudes of the elasticities of demand for polluting goods and of the substitutability between green capital and natural resources. The impact on the optimal savings rate also depends on the productivity of green capital and on the ability to adapt existing capital

  19. Green Consumerism : an Eco-Friendly Behaviour Form Through The Green Product Consumption and Green Marketing

    Directory of Open Access Journals (Sweden)

    Wiwik Handayani

    2017-09-01

    Full Text Available This research is referred to analyse the influence of consumer attitude of green product towards purchase intention. The consumer attitude of green product is a psychological tendencies that is expressed by evaluating a certain entity with some advantage or disadvantage considerations. The problem of this research is the low of cunsumer awareness to consume green product, because the lack to comprehend the importance of green product usage for health and eco-friendly. The purpose of this research is to test the influence of consumer attitude of green products towards purchase intention. Hypothesis testing using Partial Least Square (PLS. The result of analysis show that there is influence among consumer attitude of green product towards consumer purchase intention significantly.

  20. Green fluorescent protein.

    Science.gov (United States)

    Chalfie, M

    1995-10-01

    Several bioluminescent coelenterates use a secondary fluorescent protein, the green fluorescent protein (GFP), in an energy transfer reaction to produce green light. The most studied of these proteins have been the GFPs from the jellyfish Aequorea victoria and the sea pansy Renilla reniformis. Although the proteins from these organisms are not identical, they are thought to have the same chromophore, which is derived from the primary amino acid sequence of GFP. The differences are thought to be due to changes in the protein environment of the chromophore. Recent interest in these molecules has arisen from the cloning of the Aequorea gfp cDNA and the demonstration that its expression in the absence of other Aequorea proteins results in a fluorescent product. This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues. Bacterial, plant and animal (including mammalian) cells all express GFP. The heterologous expression of the gfp cDNA has also meant that it could be mutated to produce proteins with different fluorescent properties. Variants with more intense fluorescence or alterations in the excitation and emission spectra have been produced.

  1. Greening of orthopedic surgery.

    Science.gov (United States)

    Lee, Rushyuan J; Mears, Simon C

    2012-06-01

    Every year, 4 billion pounds of waste are produced by health care facilities, and the amount continues to increase annually. In response, a movement toward greening health care has been building, with a particular focus on the operating room. Between 20% and 70% of health care waste originates from a hospital's operating room, and up to 90% of operating room waste is improperly sorted and sent for costly and unneeded hazardous waste processing. Recent successful changes include segregation of hospital waste, substitution of the ubiquitous polypropylene plastic wrap used for the sterilization and handling of surgical equipment with metal cases, and the reintroduction of reusable surgical gowns. Orthopedic-related changes include the successful reprocessing and reuse of external fixators, shavers, blades, burs, and tourniquets. These changes have been shown to be environmentally and economically beneficial. Early review indicates that these changes are feasible, but a need exists for further evaluation of the effect on the operating room and flow of the surgical procedure and of the risks to the surgeons and operating room staff. Other key considerations are the effects of reprocessed and reused equipment on patient care and outcome and the role of surgeons in helping patients make informed decisions regarding surgical care. The goals of this study were to summarize the amount and types of waste produced in hospitals and operating rooms, highlight the methods of disposal used, review disposal methods that have been developed to reduce waste and improve recycling, and explore future developments in greening health care. Copyright 2012, SLACK Incorporated.

  2. Can Growth Be Green?

    Science.gov (United States)

    Gough, Ian

    2015-01-01

    This short article, based on a presentation at the London School of Economics, criticizes the common opinion that "green growth" offers a relatively painless - some even say pain-free - transition path for capitalist economies. After a brief summary of the daunting arithmetic entailed in combining fast decarbonization with continuing growth, the article advances 3 propositions. First, market-based carbon mitigation programs, such as carbon trading, cannot be sufficient and must be coupled with other policy pillars that foster transformative investment and widespread regulation. Second, a political economy of climate policy needs to draw on the lessons of comparative social policy research, which emphasizes the role of international pressures, interests, institutions, and ideas. Taking these into account gives a more realistic perspective on climate policy making in today's neoliberal world. Third, more radical policies on both consumption and production are called for, to ensure that carbon mitigation is not pursued at the expense of equity and social welfare. These include policies to restrain high-carbon luxury consumption and a transition toward shorter paid working time. The conclusion is that a realistic program of green growth will be immensely difficult and entail radical political change. © SAGE Publications 2015.

  3. Evaluation of green roof characteristics in green building assessment

    Directory of Open Access Journals (Sweden)

    Sekulić Mirjana

    2013-01-01

    Full Text Available Methodology of building evaluation based on green building characteristics is rapidly gaining momentum, mainly in foreign, but also in domestic building practice. This methodology is being carried out through different Green Building Certification Systems, which are complex evaluation mechanisms based on numerous criteria of sustainability, addressing both ecological issues, but also economic and social ones. Green roof represents one of the 'must have' features of contemporary buildings aiming to gain green label. This paradigm is based on their numerous characteristics which contribute to different aspects of building sustainability, among which are savings in energy and water consumption, but also ecological balance and quality of built environment. Criteria used for evaluation of green roof solutions and their overall contribution to the building, are integral part of all of the mentioned certification systems, but the way they are structured and formulated inside each system varies significantly, hence causing differences in evaluation results. This paper presents the analysis of green roof related criteria of three characteristic green building certification systems: LEED, BREEAM and CASBEE. These systems are chosen primarily because of the different evaluation methodology, but also because of their market prevalence and perspectives of usage in the domestic practice. Conclusions driven from these analyses and comparisons provide insight into main aspect of green roof planning and construction which are relevant for the overall building sustainability assessment.

  4. Powerful qPCR assays for the early detection of latent invaders: interdisciplinary approaches in clinical cancer research and plant pathology.

    Science.gov (United States)

    Luchi, Nicola; Capretti, Paolo; Pazzagli, Mario; Pinzani, Pamela

    2016-06-01

    Latent invaders represent the first step of disease before symptoms occur in the host. Based on recent findings, tumors are considered to be ecosystems in which cancer cells act as invasive species that interact with the native host cell species. Analogously, in plants latent fungal pathogens coevolve within symptomless host tissues. For these reasons, similar detection approaches can be used for an early diagnosis of the invasion process in both plants and humans to prevent or reduce the spread of the disease. Molecular tools based on the evaluation of nucleic acids have been developed for the specific, rapid, and early detection of human diseases. During the last decades, these techniques to assess and quantify the proliferation of latent invaders in host cells have been transferred from the medical field to different areas of scientific research, such as plant pathology. An improvement in molecular biology protocols (especially referring to qPCR assays) specifically designed and optimized for detection in host plants is therefore advisable. This work is a cross-disciplinary review discussing the use of a methodological approach that is employed within both medical and plant sciences. It provides an overview of the principal qPCR tools for the detection of latent invaders, focusing on comparisons between clinical cancer research and plant pathology, and recent advances in the early detection of latent invaders to improve prevention and control strategies.

  5. The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel

    Directory of Open Access Journals (Sweden)

    Shoshanit eOhad

    2016-02-01

    Full Text Available The emerging Microbial Source Tracking (MST methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes High Throughput Sequencing (HTS to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU analysis was then performed, from which sequences were retained for downstream qPCR marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae and other bacteria. Two assays (Av4143 and Av163 identified most of the avian fecal samples and demonstrated sensitivity values of 91% and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216 identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

  6. Isolation and Identification of Lactic Acid Bacteria from Traditional Dairy Products in Baotou and Bayannur of Midwestern Inner Mongolia and q-PCR Analysis of Predominant Species

    Science.gov (United States)

    2016-01-01

    In this study, traditional culture method and 16S rRNA gene analysis were applied to reveal the composition and diversity of lactic acid bacteria (LAB) of fermented cow milk, huruud and urum from Baotou and Bayannur of midwestern Inner Mongolia. Also, the quantitative results of dominant LAB species in three different types of dairy products from Baotou and Bayannur were gained by quantitative polymerase chain reaction (q-PCR) technology. Two hundred and two LAB strains isolated from sixty-six samples were identified and classified into four genera, namely Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, and twenty-one species and subspecies. From these isolates, Lactococcus lactis subsp. lactis (32.18%), Lactobacillus plantarum (12.38%) and Leuconosto mesenteroides (11.39%) were considered as the dominated LAB species under the condition of cultivating in MRS and M17 medium. And the q-PCR results revealed that the number of dominant species varied from samples to samples and from region to region. This study clearly shows the composition and diversity of LAB existing in fermented cow milk, huruud and urum, which could be considered as valuable resources for LAB isolation and further probiotic selection. PMID:27621691

  7. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  8. Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses

    DEFF Research Database (Denmark)

    Mærkedahl, Rasmus Baadsgaard; Frøkiær, Hanne; Lauritzen, Lotte

    2015-01-01

    -extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis. Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at -20 °C for 0, 1, or 4 months before RNA extraction....../binding solution over time and between samples stored and extracted by the two systems. Conclusions: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large......Abstract Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA...

  9. A new method for non-invasive prenatal diagnosis of Down syndrome using MeDIP real time qPCR

    Directory of Open Access Journals (Sweden)

    Philippos C. Patsalis

    2012-12-01

    Full Text Available During the last decade, the area of non-invasive prenatal diagnosis (NIPD has rapidly evolved. Several methodological approaches have been presented and demonstrated a proof of concept for the NIPD of chromosomal aneuploidies. The two most promising methods are NIPD using next generation sequencing technologies and NIPD using Methylation DNA Immunoprecipitation (MeDIP with real time qPCR. Both approaches have been validated with blind studies and have >99% accuracy. NIPD using next generation sequencing is achieved by high throughput shotgun sequencing of DNA from plasma of maternal women followed by ratio comparisons of each chromosome sequence tag density over the median tag density of all autosomes (z-score analysis. The MeDIP real time qPCR method, which is described in this review in more detail, is based on the identification of differentially methylated regions (DMRs and their use in discriminating normal from abnormal cases. More than 10,000 DMRs were identified for chromosomes 13, 18, 21, X and Y using high resolution oligo-arrays that can be potentially used for the NIPD of aneuploidies for chromosomes 13, 18, 21, X and Y. Both NIPD methods have several advantages and limitations and it is believed that they will soon be implemented in clinical practice. With the continuous advancements of genetic methodologies and technologies, we predict that within the next 10 years we will be able to provide NIPD for all common and rare genetic disorders where the molecular basis is known.

  10. Improved sample preparation for direct quantitative detection of Escherichia coli O157 in soil using qPCR without pre-enrichment.

    Science.gov (United States)

    Highmore, Callum J; Rothwell, Steve D; Keevil, Charles W

    2017-07-01

    The prominence of fresh produce as a vehicle for foodborne pathogens such as enterohaemorrhagic Escherichia coli (EHEC) O157 is rising, where disease cases can cause hospitalization and in some cases death. This rise emphasises the necessity for accurate and sensitive methods for detection of pathogens in soil, potential sources of contamination of fresh produce. The complexity of the soil matrix has previously proven prohibitive to pathogen detection via molecular methods without the use of a culture enrichment step, thereby excluding the detection of viable but non-culturable cells. Here, a sample preparation procedure to facilitate a direct qPCR assay is developed for the detection of E. coli O157 in soil, bypassing culture steps in favour of sample separation through pulsification release and filtration. In sand and peat-based compost, the method is sensitive to 10 CFU g -1 soil. When testing soils from agricultural sites, it was found that several were qPCR positive for E. coli O157 while being culture-negative, with peat-based compost possessing a concentration of 200 tir gene copies per gram. This procedure offers a rapid, quantitative assessment of the potential presence of E. coli O157 in soils which can act as a prescreen of their suitability to grow fresh produce safely. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  11. Development of a Real-Time qPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest

    Directory of Open Access Journals (Sweden)

    Robert I. Graham

    2015-08-01

    Full Text Available Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR procedure using a 5' nuclease hydrolysis (TaqMan probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV. The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.

  12. The "Green" Root Beer Laboratory

    Science.gov (United States)

    Clary, Renee; Wandersee, James

    2010-01-01

    No, your students will not be drinking green root beer for St. Patrick's Day--this "green" root beer laboratory promotes environmental awareness in the science classroom, and provides a venue for some very sound science content! While many science classrooms incorporate root beer-brewing activities, the root beer lab presented in this article has…

  13. Green accounts & day high schools

    DEFF Research Database (Denmark)

    Jørgensen, Michael Søgaard

    1997-01-01

    The arcticle presents the concept of green accounts and describes how it can be used in the daily work and the teaching at day high schools.......The arcticle presents the concept of green accounts and describes how it can be used in the daily work and the teaching at day high schools....

  14. Green grabbing debate and Madagascar

    DEFF Research Database (Denmark)

    Casse, Thorkil; Razafy, Fara Lala; Wurtzebach, Zachary

    2017-01-01

    foundations of green grabbing, the concepts of primitive accumulation and commodification of nature. Third, we compare data collected by the green grabbing scholars and conservation NGOs from the very same site in Madagascar. We conclude that rigorous post-intervention stakeholder analysis, rather than pre...

  15. Green investment: Trends and determinants

    International Nuclear Information System (INIS)

    Eyraud, Luc; Clements, Benedict; Wane, Abdoul

    2013-01-01

    This paper fills a gap in the macroeconomic literature on renewable sources of energy. It offers a definition of green investment and analyzes the trends and determinants of this investment over the last decade for 35 advanced and emerging countries. We use a new multi-country historical dataset and find that green investment has become a key driver of the energy sector and that its rapid growth is now mostly driven by China. Our econometric results suggest that green investment is boosted by economic growth, a sound financial system conducive to low interest rates, and high fuel prices. We also find that some policy interventions, such as the introduction of carbon pricing schemes or “feed-in-tariffs,” which require use of “green” energy, have a positive and significant impact on green investment. Other interventions, such as biofuel support, do not appear to be associated with higher green investment. - Highlights: • We offer a definition of green investment and review its trend since 2000. • We analyze its determinants from both theoretical and empirical perspectives. • Green investment is boosted by economic growth, interest rates, and fuel prices. • Feed-in-tariffs and carbon pricing schemes impact positively green investment

  16. News from Online: Green Chemistry

    Science.gov (United States)

    Uffelman, Erich S.

    2004-01-01

    Green chemistry closely relates to energy and environmental problems, and includes the promotion of environmental friendly products and systems within the framework of renewable resources. Various websites on green chemistry are reviewed, one of which lists the 12 commandments of this particular subject.

  17. Green tea and bone health

    Science.gov (United States)

    Osteoporosis is a major health problem in the elderly, particularly women. Epidemiological evidence has shown an association between tea consumption and the prevention of age-related bone loss in elderly women and men. Ingestion of green tea and green tea bioactive compounds may be beneficial in mit...

  18. Being Green: Managing Chemical Use

    DEFF Research Database (Denmark)

    Goodsite, Michael Evan; Warncke, Esbern; Moseholm, Lars

    2010-01-01

    The definitive, practical, go-to resource guide on helping all charities become more "green" Nonprofit Guide to Going Green is your comprehensive learning tool to guide nonprofits and NGOs towards becoming greener. A desktop reference for any charitable organization to become greener, this essent...

  19. Summary of Green Building Programs

    Energy Technology Data Exchange (ETDEWEB)

    2002-08-01

    In early 2002, the National Association of Home Builders completed a census of residential green building programs across the United States to assess differences and similarities among programs. This report catalogs different ways that builders participate in residential green building programs.

  20. Conceptual approaches to the notion "green business"

    OpenAIRE

    B. Stepanenko

    2010-01-01

    The author defines the notion "green business". Theoretical and methodological principles of functioning of this type of economic activity are reflected. Basic aspects and main development stages of green business are described. The classification of green business enterprise is revealed.

  1. Green Roofs for Stormwater Runoff Control - Abstract

    Science.gov (United States)

    This project evaluated green roofs as a stormwater management tool. Specifically, runoff quantity and quality from green and flat asphalt roofs were compared. Evapotranspiration from planted green roofs and evaporation from unplanted media roofs were also compared. The influence...

  2. Sound absorption with green materials

    Science.gov (United States)

    Trematerra, Amelia; Lombardi, Ilaria

    2017-07-01

    Green materials are a valid alternative to traditional materials that are by-products of processing oil. At the end of their useful life, green materials can be disposed of without polluting the environment. They are now being used in the construction and automotive industries. While, studies are currently being carried out in the aviation sector on the use of green materials for non-structural components of airplanes. Green materials can be used to improve the acoustic comfort inside buildings as well as mitigate reverberation, echoes effects and reduce the transmission of noise between rooms. In this paper, the acoustic measurements of the properties of green materials are reported. The absorption coefficient of samples of the materials were measured in the frequency range from 200 Hz to 2,000 Hz with an impedance tube, with the flow resistance being measured.

  3. World green electricity, sustaining investments

    International Nuclear Information System (INIS)

    Le Jannic, N.

    2013-01-01

    The contribution of the green production to the world production of electricity reached 20.2% in 2011, it means a slight increase in respect to the figure of 2010: 19.8%. Green electricity is the second source of electricity behind fossil energy (67.9%) but before nuclear power (11.7%). The decrease in nuclear power due to the Fukushima accident has automatically benefited green electricity. The figures show the importance of China, China is now the first electricity producer in the world before US and also passed US for the production of green electricity. At the world scale the production of green electricity can break down into: hydro energy (80.5%), wind energy (10.3%), biomass (6.2%), geothermal energy (1.6%) and solar energy (1.4%). The crisis has slowed down the investment in renewable energies in Europe. (A.C.)

  4. Green Fiber Bottle

    DEFF Research Database (Denmark)

    Didone, Mattia; Tosello, Guido

    The ambition of the Green Fiber Bottle (GFB) project is to manufacture a fully biodegradable bottle. Carlsberg is the intended end user, and they aim to package their beer in the new bottle. The new product is intended to replace the existing plastic and glass bottles, and thus reducing...... their impact on the environment, especially the oceans. For example, the life span of a plastic bottle in the ocean is 500 years, and during its degradation, the plastic is reduced to micro pieces, which causes the starvation of several marine animals. The new bottle is completely made from molded paper pulp......, which is a renewable resource. Nevertheless, due to food and drugs limitations, only virgin paper fibers must be employed in the production. The bottle could then be left to biodegrade in nature or enter a recycle system, along with other paper-based product. In order to contain the liquid, the bottle...

  5. VISAS AND GREEN PLATES

    CERN Multimedia

    2000-01-01

    From 3 April 2000, all questions relating to visa requests for Switzerland, France, or Russia for a member of the personnel must be addressed to Ms. Agnita Querrou (telephone 72838, office 5-2-019, e-mail Agnita.Querrou@cern.ch).The Users' Office continues to deal with requests for letters of invitation and questions concerning visas for users in EP Division.Questions relating to removals, requests for green plates, to privileges of members of the personnel and to the importation of vehicles are still dealt with by Ms Zuzana Miller (telephone 79257, office 33-1-017, e-mail Zuzana.Muller@cern.ch) and Ms Joëlle Belleman (telephone 73962, office 33-1-019, e-mail Joelle.Belleman@cern.ch).

  6. Green symbiotic cloud communications

    CERN Document Server

    Mustafa, H D; Desai, Uday B; Baveja, Brij Mohan

    2017-01-01

    This book intends to change the perception of modern day telecommunications. Communication systems, usually perceived as “dumb pipes”, carrying information / data from one point to another, are evolved into intelligently communicating smart systems. The book introduces a new field of cloud communications. The concept, theory, and architecture of this new field of cloud communications are discussed. The book lays down nine design postulates that form the basis of the development of a first of its kind cloud communication paradigm entitled Green Symbiotic Cloud Communications or GSCC. The proposed design postulates are formulated in a generic way to form the backbone for development of systems and technologies of the future. The book can be used to develop courses that serve as an essential part of graduate curriculum in computer science and electrical engineering. Such courses can be independent or part of high-level research courses. The book will also be of interest to a wide range of readers including b...

  7. Future Green Buildings

    DEFF Research Database (Denmark)

    Mathiesen, Brian Vad; Drysdale, David; Lund, Henrik

    an energy system integration perspective, heat savings, electricity savings, and user behavioural aspects as well as energy storage and household level flexibility. Many reports on green or sustainable buildings focus only on savings levels and disregard the cost of renewable energy production. Some reports......Efficient buildings are essential for an affordable Danish energy supply in 2050. The purpose of this report is to describe the contribution and role of the building sector in a 100% renewable energy future, as well as the transitions that are necessary in the building sector to support this change....... The report builds on a literature review encompassing more than 50 reports and research papers over the last 10 years and more than a two decades knowledge about the interactions between different components of the energy sector. The review has been focused on aspects such as cost-effective solutions from...

  8. Green Maritime Logistics

    DEFF Research Database (Denmark)

    Psaraftis, Harilaos N.

    2014-01-01

    Typical problems in maritime logistics include, among others, optimal ship speed, ship routing and scheduling, fleet deployment, fleet size and mix, weather routing, intermodal network design, modal split, transshipment, queuing at ports, terminal management, berth allocation, and total supply...... chain management. The traditional analysis of these problems has been in terms of cost- benefit and other optimization criteria from the point of view of the logistics provider, carrier, shipper, or other end-user. Such traditional analysis by and large either ignores environmental issues, or considers...... in the goal of greening the maritime supply chain and takes stock at models that can be used to evaluate these trade-offs. The talk is based on recent research of the author and his colleagues....

  9. Campaign Country Going Green?

    DEFF Research Database (Denmark)

    Poulsen, Bo

    2017-01-01

    This paper investigates more than 20 years of government led attempts to have Danish consumers curb their energy use. Looking at previously unexploited campaign material a number of results emerge, qualifying, the notion of Denmark as a front runner, when it comes to environmental awareness....... The first attempts to cut energy consumption came about as a direct consequence of the international oil crises of 1973-74, and for the following 15 years the government standing committee on energy savings issued a string of energy saving campaigns, fueled entirely by an appeal to common sense household...... justification. This paper finally discusses the reason for this greening of government initiated Danish energy saving campaigns, which is seen as an indirect result of the 1987 UN report, Our Common Future. The 1988 general election in Denmark led to the formation of a new center-right government coalition...

  10. Green chemistry at work

    Energy Technology Data Exchange (ETDEWEB)

    Frost, J. [Michigan State Univ., East Lansing, MI (United States)

    1994-12-31

    The 1.7 billion pounds of benzene produced each year in the US provide one measure of its utility. At the same time, there are a number of environmental reasons for avoiding the use of benzene in chemical manufacture. Perhaps most compelling: benzene is a potent carcinogen. Scrutiny of many of the chemicals derived from benzene reveals that each molecule contains at least one oxygen atom while benzene completely lacks oxygen atoms. Introduction of oxygen to make up for this lack can require processes that are environmentally problematic. One of the steps used to introduce oxygen atoms during manufacture of adipic acid, a component of Nylon 66, is responsible for 10% of the annual global increase in atmospheric nitrous oxide. This by-product is a causative agent of atmospheric ozone depletion and has been implicated in global warming. With support from EPA and the National Science Foundation, alternative manufacturing processes are being explored. By these new methods, chemicals usually created from benzene are made instead from nontoxic glucose, a component of table sugar. Unlike benzene, glucose is obtained from such renewable resources as plant starch and cellulose. ``Green`` manufacturing routes ideally should lead to chemicals that are economically competitive with chemicals produced by traditional methods. For two chemicals of roughly comparable cost, the consumer or producer can then be realistically expected to choose in favor of the chemical produced by a ``green`` process. Projections indicate that catechol and hydroquinone can be biocatalytically produced from glucose at a cost competitive with current market prices. Synthesis of chemicals from glucose using biocatalysis offers the premise of achieving fundamental environmental improvement while increasing the demand for agricultural products. In the final analysis, what is good for the environment can also be good for American agriculture.

  11. Urban Green Infrastructure: German Experience

    Directory of Open Access Journals (Sweden)

    Diana Olegovna Dushkova

    2016-06-01

    Full Text Available The paper presents a concept of urban green infrastructure and analyzes the features of its implementation in the urban development programmes of German cities. We analyzed the most shared articles devoted to the urban green infrastructure to see different approaches to definition of this term. It is based on materials of field research in the cities of Berlin and Leipzig in 2014-2015, international and national scientific publications. During the process of preparing the paper, consultations have been held with experts from scientific institutions and Administrations of Berlin and Leipzig as well as local experts from environmental organizations of both cities. Using the German cities of Berlin and Leipzig as examples, this paper identifies how the concept can be implemented in the program of urban development. It presents the main elements of green city model, which include mitigation of negative anthropogenic impact on the environment under the framework of urban sustainable development. Essential part of it is a complex ecological policy as a major necessary tool for the implementation of the green urban infrastructure concept. This ecological policy should embody not only some ecological measurements, but also a greening of all urban infrastructure elements as well as implementation of sustainable living with a greater awareness of the resources, which are used in everyday life, and development of environmental thinking among urban citizens. Urban green infrastructure is a unity of four main components: green building, green transportation, eco-friendly waste management, green transport routes and ecological corridors. Experience in the development of urban green infrastructure in Germany can be useful to improve the environmental situation in Russian cities.

  12. Going Green: The Business Case for Greening your Energy Company

    Energy Technology Data Exchange (ETDEWEB)

    Lavery, Greg

    2007-07-01

    We are all familiar with the challenges facing the energy industry: supply security, climate change, emerging cleaner technologies, retail competition, staffing, and the quest for growth. This paper demonstrates how a proactive environmentally considered ('green') corporate approach addresses these issues and unlocks four tangible areas of value addition for energy companies. Based on over a decade of experience by the author in this emerging field, this paper provides some golden rules for companies considering the green approach and showcases an Australian market leading energy company who is unlocking green value. (auth)

  13. Green Building Pro-Environment Behaviors: Are Green Users Also Green Buyers?

    Directory of Open Access Journals (Sweden)

    Xiaohuan Xie

    2017-09-01

    Full Text Available Pro-environment behaviors play a key role in advancing the development of green buildings. This study investigated the link between two green building pro-environment behaviors that require dissimilar resources: energy savings that do not require money in order to be more environmentally friendly and willingness to pay that involves economic resources including spending money in order to be more environmentally friendly. This study points out that the two pro-environment behaviors can be positively linked to each other. People who behave in an environmentally friendly manner at work would also be likely to pay an extra cost for a green building when buying a new home. The consistency of the two pro-environment behaviors can be explained by their common environmental beliefs: limits to growth and eco-crisis. The green building movement should prioritize pro-environmental behaviors and associated environmental beliefs to support green building policies, guidelines, and tools.

  14. Green materials for sustainable development

    Science.gov (United States)

    Purwasasmita, B. S.

    2017-03-01

    Sustainable development is an integrity of multidiscipline concept combining ecological, social and economic aspects to construct a liveable human living system. The sustainable development can be support through the development of green materials. Green materials offers a unique characteristic and properties including abundant in nature, less toxic, economically affordable and versatility in term of physical and chemical properties. Green materials can be applied for a numerous field in science and technology applications including for energy, building, construction and infrastructures, materials science and engineering applications and pollution management and technology. For instance, green materials can be developed as a source for energy production. Green materials including biomass-based source can be developed as a source for biodiesel and bioethanol production. Biomass-based materials also can be transformed into advanced functionalized materials for advanced bio-applications such as the transformation of chitin into chitosan which further used for biomedicine, biomaterials and tissue engineering applications. Recently, cellulose-based material and lignocellulose-based materials as a source for the developing functional materials attracted the potential prospect for biomaterials, reinforcing materials and nanotechnology. Furthermore, the development of pigment materials has gaining interest by using the green materials as a source due to their unique properties. Eventually, Indonesia as a large country with a large biodiversity can enhance the development of green material to strengthen our nation competitiveness and develop the materials technology for the future.

  15. Antioxidant effects of green tea

    Science.gov (United States)

    FORESTER, SARAH C.; LAMBERT, JOSHUA D.

    2013-01-01

    Consumption of green tea (Camellia sinensis) may provide protection against chronic diseases, including cancer. Green tea polyphenols are believed to be responsible for this cancer preventive effect, and the antioxidant activity of the green tea polyphenols has been implicated as a potential mechanism. This hypothesis has been difficult to study in vivo due to metabolism of these compounds and poor understanding of the redox environment in vivo. Green tea polyphenols can be direct antioxidants by scavenging reactive oxygen species or chelating transition metals as has been demonstrated in vitro. Alternatively, they may act indirectly by up-regulating phase II antioxidant enzymes. Evidence of this latter effect has been observed in vivo, yet more work is required to determine under which conditions these mechanisms occur. Green tea polyphenols can also be potent pro-oxidants, both in vitro and in vivo, leading to the formation of hydrogen peroxide, the hydroxyl radical, and superoxide anion. The potential role of these pro-oxidant effects in the cancer preventive activity of green tea is not well understood. The evidence for not only the antioxidant, but also pro-oxidant, properties of green tea are discussed in the present review. PMID:21538850

  16. Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

    Science.gov (United States)

    Michel, Elisa; Monfort, Clarisse; Deffrasnes, Marion; Guezenec, Stéphane; Lhomme, Emilie; Barret, Matthieu; Sicard, Delphine; Dousset, Xavier; Onno, Bernard

    2016-12-19

    In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic

  17. Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; De Keersmaecker, Sigrid C J; Delvoye, Maud; Gau, Céline; Philipp, Patrick; Roosens, Nancy H

    2015-11-11

    Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method. Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly. In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step. The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily

  18. Green Power Partnership Eligible Scope of Green Power Use

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership is a voluntary partnership program designed to reduce the environmental impact of electricity generation by promoting renewable energy. EPA requires that Partners meet GPP's vintage requirement.

  19. It's hard to be green: Reverse green value chain.

    Science.gov (United States)

    Couto, João; Tiago, Teresa; Gil, Artur; Tiago, Flávio; Faria, Sandra

    2016-08-01

    Firms have recently discovered that it is not enough to optimize internal processes and relationships with partners along the value chain to create a sustainable competitive market position. A clear customer orientation, which acknowledges that consumer buying behavior is complex and includes many elements implied in the value chain, is required. As companies offering green products are no exception to this rule, this study analyzes consumer behavior in Europe from a reserve green supply chain management perspective, using descriptive analyses and a structural equation model, with data collected by Flash Barometer comprising 26,573 responses from 28 European countries. The results suggest that European consumers are conscious of the green concept, but are not willing to buy or pay more for these products since the value is unclear. Companies offering green products must therefore rethink their strategies, especially in terms of value proposition, communication strategies, and eco-labeling. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Green's functions in quantum physics

    CERN Document Server

    Economou, Eleftherios N

    2006-01-01

    The main part of this book is devoted to the simplest kind of Green's functions, namely the solutions of linear differential equations with a -function source. It is shown that these familiar Green's functions are a powerful tool for obtaining relatively simple and general solutions of basic problems such as scattering and bound-level information. The bound-level treatment gives a clear physical understanding of "difficult" questions such as superconductivity, the Kondo effect, and, to a lesser degree, disorder-induced localization. The more advanced subject of many-body Green's functions is presented in the last part of the book.

  1. Green Energy - Fiction and Reality

    Science.gov (United States)

    Cranberg, Lawrence

    2009-03-01

    The term ``Green Energy'' has been popularized to refer to energy sources that do not depend on fossil fuels. The oldest truly ``green'' energy source is wood fuel derived by photosynthesis for thermal comfort in the cold season. Thermal energy from the combustion of wood for personal heating in our 41 million fireplaces has greatly declined, due to an ``Anti-fireplace Hoax'' (1) that fireplaces are ``energy counterproductive.'' Physicists have a special obligation to address the problem that our major true source of ``Green Energy'' is widely misrepresented and neglected. 1. L. Cranberg, The Physics Teacher, Letter, January,l989

  2. Catalysis and sustainable (green) chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Centi, Gabriele; Perathoner, Siglinda [Dipartimento di Chimica Industriale ed Ingegneria dei Materiali, University of Messina, Salita Sperone 31, 98166 Messina (Italy)

    2003-01-15

    Catalysis is a key technology to achieve the objectives of sustainable (green) chemistry. After introducing the concepts of sustainable (green) chemistry and a brief assessment of new sustainable chemical technologies, the relationship between catalysis and sustainable (green) chemistry is discussed and illustrated via an analysis of some selected and relevant examples. Emphasis is also given to the concept of catalytic technologies for scaling-down chemical processes, in order to develop sustainable production processes which reduce the impact on the environment to an acceptable level that allows self-depuration processes of the living environment.

  3. A Green Corridor Balanced Scorecard

    Directory of Open Access Journals (Sweden)

    Prause Gunnar

    2014-12-01

    Full Text Available Green transport corridors represent trans-shipment routes with a concentration of freight traffic between major hubs and long distances of transport marked by reduced environmental and climate impact. Important characteristics of green corridors are their network structures, their transnational character and their high involvement of public and private stakeholders, including political level requiring new governance models. Network-oriented controlling of green transport corridors require new concepts and instruments concentrating on multi-dimensional evaluation of collective strategies and processes in an international environment with a focus on cross-company aspects.

  4. Green roofs: potential at LANL

    Energy Technology Data Exchange (ETDEWEB)

    Pacheco, Elena M [Los Alamos National Laboratory

    2009-01-01

    Green roofs, roof systems that support vegetation, are rapidly becoming one of the most popular sustainable methods to combat urban environmental problems in North America. An extensive list of literature has been published in the past three decades recording the ecological benefits of green roofs; and now those benefits have been measured in enumerated data as a means to analyze the costs and returns of green roof technology. Most recently several studies have made substantial progress quantifying the monetary savings associated with storm water mitigation, the lessoning of the Urban Heat Island, and reduction of building cooling demands due to the implementation of green roof systems. Like any natural vegetation, a green roof is capable of absorbing the precipitation that falls on it. This capability has shown to significantly decrease the amount of storm water runoff produced by buildings as well as slow the rate at which runoff is dispensed. As a result of this reduction in volume and velocity, storm drains and sewage systems are relieved of any excess stress they might experience in a storm. For many municipalities and private building owners, any increase in storm water mitigation can result in major tax incentives and revenue that does not have to be spent on extra water treatments. Along with absorption of water, vegetation on green roofs is also capable of transpiration, the process by which moisture is evaporated into the air to cool ambient temperatures. This natural process aims to minimize the Urban Heat Island Effect, a phenomenon brought on by the dark and paved surfaces that increases air temperatures in urban cores. As the sun distributes solar radiation over a city's area, dark surfaces such as bitumen rooftops absorb solar rays and their heat. That heat is later released during the evening hours and the ambient temperatures do not cool as they normally would, creating an island of constant heat. Such excessively high temperatures induce heat

  5. Progress report on Green Deals 2013; Voortgangsrapportage Green Deals 2013

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2013-11-15

    In the Dutch governmental coalition agreement the Green Deal approach was announced in the autumn of 2010. The focus of the Green Deals is for people and companies to develop sustainable initiatives that contribute to economic growth. The Green Deal approach started with the theme energy, but has been extended with the themes biobased economy, climate, resources, buildings, food, mobility, water and biological diversity. This progress report provides an overview of the deals that this bottom-up approach has yielded. The report also provides information on the progress of the deals and the interim results of the approach and the individual deals. Also attention is paid to how the 146 Green Deals score on innovation and entrepreneurship [Dutch] In het regeerakkoord van het kabinet is in het najaar van 2010 de Green Deal-aanpak aangekondigd. Centraal in de aanpak staat dat mensen en bedrijven zoveel mogelijk ruimte krijgen voor eigen duurzame initiatieven die bijdragen aan economische groei. De aanpak is gestart vanuit het thema energie, maar beslaat inmiddels ook de thema's biobased economy, klimaat, grondstoffen, bouw, voedsel, mobiliteit, water en biodiversiteit. Deze voortgangsrapportage geeft een overzicht van de deals die deze bottom-up aanpak heeft opgeleverd. De rapportage informeert bovendien over de voortgang van de deals en over de tussentijdse resultaten van zowel de aanpak als de afzonderlijke deals. Ook wordt gekeken hoe de 146 Green Deals scoren op innovatief vermogen en ondernemerschap.

  6. Properties of the reverse transcription reaction in mRNA quantification

    DEFF Research Database (Denmark)

    Ståhlberg, Anders; Håkansson, Joakim; Xian, Xiaojie

    2004-01-01

    BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure...... the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. RESULTS: Experimental variation in reverse transcription-QPCR (RT...... are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction....

  7. Renewing our green spaces

    CERN Document Server

    CERN Bulletin

    2010-01-01

    CERN’s poplars were planted 50 years ago to soak up the surplus water in the ground beneath the Laboratory’s sites. Now that they have reached the end of their life-cycle, some of the poplars are in danger of falling or losing their dead branches. On Saturday, 17 April, as part of the campaign launched in February at Prévessin, work will start on replacing the poplars on central areas of the Meyrin site.   Replanting plan for the Cedars and kindergarten carparks. In July 2009, CERN was awarded an environmental label for its protection of rare flower species and the natural landscaping approach it has taken at its Meyrin site. The Laboratory has a very strict environmental policy: for years those in charge of CERN's green areas have given priority to natural management methods and have avoided the use of pesticides. In addition, patches of land are left unmowed in spring, allowing the local flora—especially the orchids—to grow naturally. Since l...

  8. Greening the Judiciary

    Directory of Open Access Journals (Sweden)

    M Kidd

    2006-12-01

    Full Text Available Much of South Africa’s environmental law is relatively new. Most of South Africa’s judges received their formal legal educations before promulgation of the major part of our environmental law and almost certainly before environmental law was taught at universities. In recent years, there have been increasing instances of cases involving environmental matters coming to the courts. How are judges performing in these cases? It would appear that the judges’ performance is rather 'chequered' in environmental cases, which suggests that the judiciary needs to become more attuned to environmental law. I call this process, for purposes of this note, ‘greening the judiciary’. What I mean by this is not that judges must decide all environmental cases in a way that favours the environment, but that they must correctly consider, interpret and apply the relevant environmental law, and give environmental considerations appropriate deliberation. This note aims to identify, in admittedly somewhat general terms, the current state of environmental decision-making by judges and to suggest what needs to happen for such decisions to be improved.

  9. Comparison of bacterial culture and qPCR testing of rectal and pen floor samples as diagnostic approaches to detect enterotoxic Escherichia coli in nursery pigs

    DEFF Research Database (Denmark)

    Weber, N. R.; Nielsen, J. P.; Hjulsager, Charlotte Kristiane

    2017-01-01

    Enterotoxigenic E. coli (ETEC) are a major cause of diarrhoea in weaned pigs. The objective of this study was to evaluate the agreement at pen level among three different diagnostic approaches for the detection of ETEC in groups of nursery pigs with diarrhoea. The diagnostic approaches used were......: bacterial culturing of faecal samples from three pigs (per pen) with clinical diarrhoea and subsequent testing for virulence genes in E. coli isolates; bacterial culturing of pen floor samples and subsequent testing for virulence genes in E. coli isolates; qPCR testing of pen floor samples in order...... to determine the quantity of F18 and F4 genes. The study was carried out in three Danish pig herds and included 31 pens with a pen-level diarrhoea prevalence of > 25%, as well as samples from 93 diarrhoeic nursery pigs from these pens. All E. coli isolates were analysed by PCR and classified as ETEC when genes...

  10. Comparison of individual and pooled samples for quantification of antimicrobial resistance genes in swine feces by high-throughput qPCR

    DEFF Research Database (Denmark)

    Clasen, Julie; Mellerup, Anders; Olsen, J. E.

    2015-01-01

    There is a considerable societal interest in the careful monitoring of antimicrobial resistance (AMR) levels in human and animal populations. Sampling and data analysis can be both costly and time consuming. Optimization of sample pooling procedures is therefore important to reduce costs...... and analysis times. The objective of this study was to estimate how many individual fecal samples are needed to pool to get a representative sample for quantification of AMR-genes in a Danish pig herd. 20 individual fecal samples were collected from one section in a Danish pig herd. One to five rectal fecal...... samples were taken from each pen with respect to the number of pigs in the pen. A total of 48 pools were made of increasing number of individual samples. The levels of 9 different AMR-genes were quantified using dynamic qPCR arrays on the BioMark HD system(Fluidigm®).DNA was extracted using the Maxwell...

  11. Validation of GUt Low Density Array (GULDA), a novel qPCR approach to the study of the intestinal microbial ecosystem

    DEFF Research Database (Denmark)

    Bergström, Anders; Licht, Tine Rask; Bahl, Martin Iain

    Causal relationships between the vast numbers of bacterial species present in the human intestines contain a lot of potential information on the regulation of the gut in the healthy as well as in diseased states. Based on the hypothesis that the human gut microbiota constitutes a dynamic ecosyste...... one standard qPCR program is used for ~40 primer sets, validation is important. We present here strategies involved in verification of GULDA as a valid tool for analysis of the human gut microbiota......., interesting correlations between the presences of the given species should exist at any time. In order to analyze this, we have developed GULDA, a cheap, flexible, reliable and high throughput qPCR-based gut low-density array (GULDA), which simultaneously gives the quantities of approximately 40 different...

  12. Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Serrano Oscar K

    2008-10-01

    Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

  13. Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase.

    Science.gov (United States)

    Neal-McKinney, Jason M; Liu, Kun C; Jinneman, Karen C; Wu, Wen-Hsin; Rice, Daniel H

    2018-01-01

    Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan-Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase ( cst ) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

  14. Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase

    Directory of Open Access Journals (Sweden)

    Jason M. Neal-McKinney

    2018-03-01

    Full Text Available Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan–Barre syndrome (GBS. GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR method and use whole genome sequencing data to detect the Campylobactersialyltransferase (cst genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

  15. Incorporating Green Infrastructure into TMDLs

    Science.gov (United States)

    The fact sheet provides examples of how some states describe green infrastructure and low impact development activities in their TMDL implementation sections to address stormwater-source impaired waters.

  16. The challenges of green nanotechnology

    Directory of Open Access Journals (Sweden)

    Miguel de la Guardia

    2014-03-01

    Full Text Available Nanomaterials have great impacts on life sciences; however,these advanced materials may induce inadvertant consequences.Thus, this editorial will highlight the futuristic challenges in green nanotechnology.

  17. Green Building and School Construction.

    Science.gov (United States)

    DiNola, Ralph; Guerra, Jerry

    2002-01-01

    Discusses the benefits of green, or high-performance, buildings, such as health and comfort, cost effectiveness, and sustainability. Explores the barriers to their use by schools--most notably cost. Offers suggestions on overcoming these barriers. (EV)

  18. Green Power Partnership Eligible Organizations

    Science.gov (United States)

    The U.S. EPA's Green Power Partnership is a voluntary partnership program designed to reduce the environmental impact of electricity generation by promoting renewable energy. Many different types of organizations are eligible to become Partners.

  19. Green Infrastructure for Arid Communities

    Science.gov (United States)

    how green infrastructure practices and the many associated benefits can be effective not only in wetter climates, but also for those communities in arid and semi-arid regions around the nation that have different precipitation patterns

  20. Promoting sustainability through green chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Kirchhoff, Mary M. [American Chemical Society, 1155 Sixteenth Street, NW, Washington, DC 20036 (United States)

    2005-06-15

    Green chemistry is an important tool in achieving sustainability. The implementation of green chemistry, the design of chemical products and processes that reduce or eliminate the use and generation of hazardous substances, is essential if the expanding global population is to enjoy an increased standard of living without having a negative impact on the health of the planet. Cleaner technologies will allow the chemical enterprise to provide society with the goods and services on which it depends in an environmentally responsible manner. Green chemistry provides solutions to such global challenges as climate change, sustainable agriculture, energy, toxics in the environment, and the depletion of natural resources. A collaborative effort by industry, academia, and government is needed to promote the adoption of the green chemistry technologies necessary to achieve a sustainable society.

  1. Green Tea and Breast Cancer

    Science.gov (United States)

    Wu, Anna H; Butler, Lesley M

    2014-01-01

    The identification of modifiable lifestyle factors that could reduce the risk of breast cancer is a research priority. Despite the enormous chemo preventive potential of green tea and compelling evidence from animal studies, its role in breast cancer development in humans is still unclear. Part of the uncertainty is related to the relatively small number of epidemiological studies on green tea and breast cancer and that the overall results from case-control studies and prospective cohort studies are discordant. In addition, the mechanisms by which green tea intake may influence risk of breast cancer in humans remains not well studied. We review the human studies that have evaluated the relationship between green tea intake and four biomarkers (sex steroid hormones, mammographic density, insulin-like growth factor, adiponectin) that are believed to be important in breast cancer development. Results from these biomarker studies are also inconclusive. Limitations of human studies and areas of further investigations are discussed. PMID:21538855

  2. Remote sensing: A green illusion

    Science.gov (United States)

    Soudani, Kamel; François, Christophe

    2014-02-01

    An analysis reveals that satellite-observed increases in canopy greenness during dry seasons, which were previously interpreted as positive responses of Amazon forests to more sunlight, are in fact an optical artefact. See Letter p.221

  3. Greening transportation at the border.

    Science.gov (United States)

    2011-01-01

    On February 2324, 2011, Federal agencies from the United : States, Canada, and Mexico sponsored a workshop in San : Diego, California, to discuss opportunities for improvement : regarding the greening of transportation at the borders.1 : The works...

  4. Advances in Green Organic Sonochemistry.

    Science.gov (United States)

    Draye, Micheline; Kardos, Nathalie

    2016-10-01

    Over the past 15 years, sustainable chemistry has emerged as a new paradigm in the development of chemistry. In the field of organic synthesis, green chemistry rhymes with relevant choice of starting materials, atom economy, methodologies that minimize the number of chemical steps, appropriate use of benign solvents and reagents, efficient strategies for product isolation and purification and energy minimization. In that context, unconventional methods, and especially ultrasound, can be a fine addition towards achieving these green requirements. Undoubtedly, sonochemistry is considered as being one of the most promising green chemical methods (Cravotto et al. Catal Commun 63: 2-9, 2015). This review is devoted to the most striking results obtained in green organic sonochemistry between 2006 and 2016. Furthermore, among catalytic transformations, oxidation reactions are the most polluting reactions in the chemical industry; thus, we have focused a part of our review on the very promising catalytic activity of ultrasound for oxidative purposes.

  5. Estimates of Green potentials. Applications

    International Nuclear Information System (INIS)

    Danchenko, V I

    2003-01-01

    Optimal Cartan-type covers by hyperbolic discs of carriers of Green α-potentials are obtained in a simply connected domain in the complex plane and estimates of the potentials outside the carriers are presented. These results are applied to problems on the separation of singularities of analytic and harmonic functions. For instance, uniform and integral estimates in terms of Green capacities of components of meromorphic functions are obtained

  6. Green Cloud on the Horizon

    Science.gov (United States)

    Ali, Mufajjul

    This paper proposes a Green Cloud model for mobile Cloud computing. The proposed model leverage on the current trend of IaaS (Infrastructure as a Service), PaaS (Platform as a Service) and SaaS (Software as a Service), and look at new paradigm called "Network as a Service" (NaaS). The Green Cloud model proposes various Telco's revenue generating streams and services with the CaaS (Cloud as a Service) for the near future.

  7. GREEN MARKETING AND GLOBAL SCENARIOS‎

    OpenAIRE

    Dr. Jessy George; R. Jith

    2017-01-01

    Green Marketing must satisfy two objectives Improved Environmental Quality and Customer Satisfaction. Although no consumer product has a zero impact on the environment, in business, the terms "green product" and "environmental product" are used commonly to describe those that strive to protect or enhance the natural environment by conserving energy and/or resources and reducing or eliminating use of toxic agents, pollution, and waste. The present study has made an attempt to describe why the ...

  8. Green Construction in Building Renovation

    Directory of Open Access Journals (Sweden)

    Ksit Barbara

    2016-06-01

    Full Text Available Modern materials and construction solutions draw more and more attention to ecology and building certification. Among the criteria appearing in revitalization, an important element is bringing plants back into heavily urbanized areas. In its natural form, this is not possible to carry out everywhere, often requiring large amounts of space. Nowadays, however, there are a number of green roofs and green wall systems, allowing “greener” construction without making significant changes in the urban environment.

  9. Stockholm: green economy leader report

    OpenAIRE

    Graham Floater; Philipp Rode; Dimitri Zenghelis

    2013-01-01

    Stockholm is a leading city for green economic growth. Despite the global downturn, the city’s low carbon economy remains highly competitive and well positioned for driving sustained growth in the medium to long term. This report, produced in partnership with the City of Stockholm, shows that Stockholm took early action to build a green economy – unlike most cities, environmental policies have been important to Stockholm for over 40 years. At the same time, early infrastructure investment suc...

  10. Green corridors in freight logistics

    DEFF Research Database (Denmark)

    Panagakos, George

    The subject of this thesis is ‘green corridors,’ a European concept denoting a concentration of freight traffic between major hubs and by relatively long distances. Since their inception in 2007, green corridors have gained popularity as a policy tool that enhances the overall environmental susta...... of stakeholder data on the selected ‘basket’ of transport services would then enable monitoring progress towards meeting the objectives that corridor management has set....

  11. How to define green adjuvants.

    Science.gov (United States)

    Beck, Bert; Steurbaut, Walter; Spanoghe, Pieter

    2012-08-01

    The concept 'green adjuvants' is difficult to define. This paper formulates an answer based on two approaches. Starting from the Organisation for Economic Cooperation and Development (OECD) definition for green chemistry, production-based and environmental-impact-based definitions for green adjuvants are proposed. According to the production-based approach, adjuvants are defined as green if they are manufactured using renewable raw materials as much as possible while making efficient use of energy, preferably renewable energy. According to the environmental impact approach, adjuvants are defined as green (1) if they have a low human and environmental impact, (2) if they do not increase active ingredient environmental mobility and/or toxicity to humans and non-target organisms, (3) if they do not increase the exposure to these active substances and (4) if they lower the impact of formulated pesticides by enhancing the performance of active ingredients, thus potentially lowering the required dosage of active ingredients. Based on both approaches, a tentative definition for 'green adjuvants' is given, and future research and legislation directions are set out. Copyright © 2012 Society of Chemical Industry.

  12. GREEN CHEMISTRY: NEW CHEMICAL PHILOSOPHY

    Directory of Open Access Journals (Sweden)

    F. A. Tykhomirova

    2015-11-01

    Full Text Available The review deals with the principles and guidelines of “Green chemistry” in comparison with the philosophy of nanotechnology. Modern philosophy and methodology of science research focus is on the process of the growth of scientific knowledge. Modern chemistry is complex, hierarchical, multilevel and multidimensional system. Philosophy of nanotechnology relies heavily on the value of scientism and the idea of domination of man over nature , there is an apology of human intervention in nature. “Green chemistry” is called “new thinking”of chemistry, philosophy of modern chemical research. The chemicals and processes in accordance with the principles of “Green chemistry” are considered not only in terms of production of substances and materials with desired properties, but also taking into account the consequences for the environment. In the “Green chemistry” created image of the “ideal customer” – he uses a minimum number of products understands the need to preserve the environment. Ideological landmark “Green chemistry” – co-evolution of man and nature, preservation of the biosphere. It emphasized the need to implement the ideology of “Green chemistry” in the training of future specialists.

  13. Green ergonomics: definition and scope.

    Science.gov (United States)

    Thatcher, Andrew

    2013-01-01

    This paper demonstrates that the goals of ergonomics (i.e. effectiveness, efficiency, health, safety and usability) are closely aligned with the goals of design for environmental sustainability. In this paper, the term 'green ergonomics' is conceptualised to specifically describe ergonomics interventions with a pro-nature emphasis. Green ergonomics is focused on the bi-directional connections between human systems and nature. This involves looking at (1) how ergonomics design and evaluation might be used to conserve, preserve, and restore nature and (2) how ecosystem services might be harnessed to facilitate the improved wellbeing and effectiveness of human systems. The paper proposes the scope of green ergonomics based on these bi-directional relationships in the areas of the design of low resource systems and products, the design of green jobs, and the design for behaviour change. Suggestions for further work in the green ergonomics domain are also made. Given the enormous environmental challenges facing modern industrial society, this paper encourages ergonomics science to embrace a pro-nature understanding of work design and research. This paper sets out the role for green ergonomics based on an appreciation of the human-nature connections that have been integrated with our understanding of ergonomics science and practice.

  14. Consumer perceptions of green power

    International Nuclear Information System (INIS)

    Rowlands, I.; Parker, P.; Scott, D.

    2001-01-01

    The relationship between consumer perceptions of the environmental impact of different energy sources and their willingness to pay a premium for green electricity was examined using the ANOVA analysis of variance and the chi-square test procedures. Since green power producers can include several energy resources in their offerings, it is important to understand the preferences of their customers. A survey sent to 480 residents in the Waterloo region of southern Ontario showed that when asked about purchasing nuclear, large scale hydropower or natural gas out of a choice of 11 energy sources, there was a wide discrepancy between those who stated a willingness to pay a large premium for green power and those who stated a willingness to pay only a small, or no, premium for green power. It was determined that these 3 energy resources were not popular among the most environmentally inclined portion of the consumer market. It was noted however that willingness to pay should be interpreted with caution because peoples' stated intentions to pay a premium for green power do not necessarily translate into action once they have the opportunity to buy power from renewable energy sources. However, marketing strategies could be used to improve the uptake of green power by consumers. The study suggests that landfill gas, is probably preferred to large-scale hydro. It was also suggested that the relative position of biomass, natural gas, garbage and nuclear power should be examined more closely. 29 refs., 3 tabs., 3 figs

  15. Enhancing Green Absorptive Capacity, Green Dynamic Capacities and Green Service Innovation to Improve Firm Performance: An Analysis of Structural Equation Modeling (SEM

    Directory of Open Access Journals (Sweden)

    Yu-Shan Chen

    2015-11-01

    Full Text Available This study discusses the influences of green absorptive capacity, green dynamic capacities, and green service innovation on firm performance. In order to fill the research gap, this study proposes the concept of green service innovation. The results are as follows: First, this study finds that green absorptive capacity has positive effects on green dynamic capacities, green service innovation, and firm performance. Second, this study points out that green dynamic capacities have positive effects on green service innovation and firm performance. Third, this study observes that green dynamic capacities and green service innovation intercede the positive connection between green absorptive capacity and firm performance.

  16. Frugal Innovation and Green Business Models

    DEFF Research Database (Denmark)

    Andersen, Maj Munch

    2015-01-01

    The literature on ‘green business models’ is rapidly developing these years. This paper suggests that much existing work on green business models lacks a deeper theoretical understanding of eco-innovation and the green economy. The paper forwards an evolutionary economic perspective on green...... business models. This perspective departs in important ways from other approaches to green business models the implications of which are sought clarified and discussed in the paper. The paper argues for the need to link up green business model innovation to aggregate green economic change. The paper posits...... that the greening of the economy has reached such a stage of maturity where a generic ‘green business model’ is apparent. The paper points to eight characteristics of eco-innovation on the basis of which key changes to the business model are identified and schematised for the different stages of the green economic...

  17. The skeptical green consumer revisited: testing the relationship between green consumerism and skepticism toward advertising

    NARCIS (Netherlands)

    Matthes, J.; Wonneberger, A.

    2014-01-01

    This article revisits the widely believed notion of the skeptical green consumer, in other words, that green consumers tend to distrust green advertising. Study 1, a survey of U.S. consumers, found no positive relationship between green consumerism and general ad skepticism. However, green

  18. Green Power Initiative

    Energy Technology Data Exchange (ETDEWEB)

    Butler, Patrick Barry [Univ. of Iowa, Iowa City, IA (United States)

    2013-01-28

    National energy policy supports the gathering of more detailed and authoritative data on the introduction of renewable bio-based fuels into new and existing district energy systems via the application of biomass gasification. The University of Iowa developed a biomass-fueled, university-scale steam generation system based on biomass gasification technologies. The system serves as a state-of-the-art research and educational facility in the emerging application of gasification in steam generation. The facility, which includes a smaller down-draft gasifier and a larger multi-stage biomass boiler, was designed to operate primarily on wood-based fuels, but has provisions for testing other biomass fuel sources produced within a 100-mile radius, providing enough flexibility to meet the fluctuating local supply of biomass from industry and Midwest agriculture. The equipment was installed in an existing, staffed facility. The down-draft gasifier unit is operated by College of Engineering staff and students, under the direct technical supervision of qualified Utilities plant staff. The Green Power Initiative also includes a substantial, innovative educational component. In addition to an onsite, graduate-level research program in biomass fuels, the investigators have integrated undergraduate and graduate level teaching – through classroom studies and experiential learning – and applied research into a biomass-based, university-scale, functioning power plant. University of Iowa is unique in that it currently has multiple renewable energy technologies deployed, including significant biomass combustion (oat hulls) at its Main Power Plant and a new reciprocating engine based renewable district energy system. This project complements and supports the national energy policy and State of Iowa initiatives in ethanol and biodiesel. Byproducts of ethanol and biodiesel processes (distiller grains) as well as industry residues (oat hulls, wood chips, construction and demolition

  19. A green hydrogen economy

    International Nuclear Information System (INIS)

    Clark, Woodrow W.; Rifkin, Jeremy

    2006-01-01

    This paper is the result of over a dozen scholars and practitioners who strongly felt that a hydrogen economy and hence the future is closer than some American politicians and bureaucrats state. Moreover, when seen internationally, there is strong evidence, the most recent and obvious ones are the proliferation of hybrid vehicles, that for any nation-state to be energy independent it must seek a renewable or green hydrogen future in the near term. The State of California has once again taken the lead in this effort for both an energy-independent future and one linked strongly to the hydrogen economy. Then why a hydrogen economy in the first instance? The fact is that hydrogen most likely will not be used for refueling of vehicles in the near term. The number of vehicles to make hydrogen commercially viable will not be in the mass market by almost all estimates until 2010. However, it is less than a decade away. The time frame is NOT 30-40 years as some argue. The hydrogen economy needs trained people, new ventures and public-private partnerships now. The paper points out how the concerns of today, including higher costs and technologies under development, can be turned into opportunities for both the public and private sectors. It was not too long ago that the size of a mobile phone was that of a briefcase, and then almost 10 years ago, the size of a shoe box. Today, they are not only the size of a man's wallet but also often given away free to consumers who subscribe or contract for wireless services. While hydrogen may not follow this technological commercialization exactly, it certainly will be on a parallel path. International events and local or regional security dictate that the time for a hydrogen must be close at hand

  20. Green Streets, Green Jobs, Green Towns (G3) Guides and Publications

    Science.gov (United States)

    This page provides links to guides and resources for: policy, regulations, and incentives; planning and design; construction, operation, and maintenance; financing and economic benefits; and, green jobs and training.