Contribution of the qPCR for the Diagnosis of Pneumocystosis

PubMed Central

Issa, Nahema; Gabriel, Frederic; Baulier, Gildas; Accoceberry, Isabelle; Mourissoux, Gaelle; Guisset, Olivier; Camou, Fabrice

2017-01-01

Abstract Background Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal respiratory infection. The incidence of PCP has decreased among HIV patients, however among non HIV-negative patients on immunosuppressive drugs; an increase in incidence is noted. In this population, the diagnosis of PCP is difficult because the clinical presentation is atypical and the direct examination (DE) of the respiratory secretions is often negative. In this context, detection of Pneumocystis jirovecii DNA in respiratory secretions by real-time quantitative chain reaction (qPCR) should be usefull. Methods In order to evaluate the usefulness of qPCR, all patients hospitalized in medicine or intensive care unit (ICU) in a university hospital and having a positive qPCR in respiratory secretions were included in a retrospective study conducted between 2013 and 2016. Based on clinical data, respiratory secretions, imaging and treatment, patients were classified into three groups: certain PCP, possible, or colonization, irrespective of the value of qPCR. Results One hundred and fifty patients, including 38 infected with HIV, were included: 75 in medicine and 75 in intensive care. Ninety patients (60%) had bronchoalveolar lavage. The diagnosis of PCP was considered certain or possible for 52 and 77 patients respectively and rejected (colonization) for 21 patients. DE was negative for 78% of non-HIV patients and 29% of HIV patients. Among the 129 patients with PCP, the hospital mortality was 35.9% in ICU and 21.5% in medicine. The median value of qPCR was 76,650 copies/mL among patients with PCP and 3,220 copies/mL among colonized patients (P < 0.001) with no significant difference in type of respiratory specimen or place of hospitalization. The optimal threshold value of qPCR determined from the ROC curve was 10,100 copies/mL with a sensitivity of 76.6% and a specificity of 86%. Specificity was 100% at the threshold of 59,250 copies/mL. Conclusion If qPCR alone is imperfect

Mathematics of quantitative kinetic PCR and the application of standard curves.

PubMed

Rutledge, R G; Côté, C

2003-08-15

Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.

The standard centrifuge method accurately measures vulnerability curves of long-vesselled olive stems.

PubMed

Hacke, Uwe G; Venturas, Martin D; MacKinnon, Evan D; Jacobsen, Anna L; Sperry, John S; Pratt, R Brandon

2015-01-01

The standard centrifuge method has been frequently used to measure vulnerability to xylem cavitation. This method has recently been questioned. It was hypothesized that open vessels lead to exponential vulnerability curves, which were thought to be indicative of measurement artifact. We tested this hypothesis in stems of olive (Olea europea) because its long vessels were recently claimed to produce a centrifuge artifact. We evaluated three predictions that followed from the open vessel artifact hypothesis: shorter stems, with more open vessels, would be more vulnerable than longer stems; standard centrifuge-based curves would be more vulnerable than dehydration-based curves; and open vessels would cause an exponential shape of centrifuge-based curves. Experimental evidence did not support these predictions. Centrifuge curves did not vary when the proportion of open vessels was altered. Centrifuge and dehydration curves were similar. At highly negative xylem pressure, centrifuge-based curves slightly overestimated vulnerability compared to the dehydration curve. This divergence was eliminated by centrifuging each stem only once. The standard centrifuge method produced accurate curves of samples containing open vessels, supporting the validity of this technique and confirming its utility in understanding plant hydraulics. Seven recommendations for avoiding artefacts and standardizing vulnerability curve methodology are provided. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

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

PubMed

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

2016-05-28

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.

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

PubMed Central

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

Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring.

PubMed

Templar, Alexander; Woodhouse, Stefan; Keshavarz-Moore, Eli; Nesbeth, Darren N

2016-08-01

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50pg (4.91×10(3) copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300pg gDNA (2.95×10(4) copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600=80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600=80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children.

PubMed

Olwagen, Courtney P; Adrian, Peter V; Madhi, Shabir A

2017-07-05

S. pneumoniae is a common colonizer of the human nasopharynx in high income and low-middle income countries. Due to limitations of standard culture methods, the prevalence of concurrent colonization with multiple serotypes is unclear. We evaluated the use of multiplex quantitative PCR (qPCR) to detect multiple pneumococcal serotypes/group colonization in archived nasopharyngeal swabs of pneumococcal conjugate vaccine naive children who had previously been investigated by traditional culture methods. Overall the detection of pneumococcal colonization was higher by qPCR (82%) compared to standard culture (71%; p < 0.001), with a high concordance (kappa = 0.73) of serotypes/groups identified by culture also being identified by qPCR. Also, qPCR was more sensitive in detecting multiple serotype/groups among colonized cases (28.7%) compared to culture (4.5%; p < 0.001). Of the additional serotypes detected only by qPCR, the majority were of lower density (<10 4 CFU/ml) than the dominant colonizing serotype, with serotype/group 6A/B, 19B/F and 23F being the highest density colonizers, followed by serotype 5 and serogroup 9A/L/N/V being the most common second and third colonizers respectively. The ability of qPCR to detect multiple pneumococcal serotypes at a low carriage density might provide better insight into underlying mechanism for changes in serotype colonization in PCV vaccinated children.

Rapid diagnosis of common deletional α-thalassemia in the Chinese population by qPCR based on identical primer homologous fragments.

PubMed

Long, Ju

2016-05-01

In China, -(SEA), -α(3.7) and -α(4.2) are common deletional α-thalassemia alleles. Gap-PCR is the currently used detection method for these alleles, whose disadvantages include time-consuming procedure and increased potential for PCR product contamination. Therefore, this detection method needs to be improved. Based on identical-primer homologous fragments, a qPCR system was developed for deletional α-thalassemia genotyping, which was composed of a group of quantitatively-related primers and their corresponding probes plus two groups of qualitatively-related primers and their corresponding probes. In order to verify the accuracy of the qPCR system, known genotype samples and random samples are employed. The standard curve result demonstrated that designed primers and probes all yielded good amplification efficiency. In the tests of known genotype samples and random samples, sample detection results were consistent with verification results. In detecting αα, -(SEA), -α(3.7) and -α(4.2) alleles, deletional α-thalassemia alleles are accurately detected by this method. In addition, this method is provided with a wider detection range, greater speed and reduced PCR product contamination risk when compared with current common gap-PCR detection reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

Improving qPCR telomere length assays: Controlling for well position effects increases statistical power.

PubMed

Eisenberg, Dan T A; Kuzawa, Christopher W; Hayes, M Geoffrey

2015-01-01

Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although, easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements. qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age, and between mother and offspring are examined. First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error as compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity in our study. Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, rerunning analyses of previous results with well position correction could serve as an independent test of the validity of these results. © 2015 Wiley Periodicals, Inc.

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

PubMed

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.

chipPCR: an R package to pre-process raw data of amplification curves.

PubMed

Rödiger, Stefan; Burdukiewicz, Michał; Schierack, Peter

2015-09-01

Both the quantitative real-time polymerase chain reaction (qPCR) and quantitative isothermal amplification (qIA) are standard methods for nucleic acid quantification. Numerous real-time read-out technologies have been developed. Despite the continuous interest in amplification-based techniques, there are only few tools for pre-processing of amplification data. However, a transparent tool for precise control of raw data is indispensable in several scenarios, for example, during the development of new instruments. chipPCR is an R: package for the pre-processing and quality analysis of raw data of amplification curves. The package takes advantage of R: 's S4 object model and offers an extensible environment. chipPCR contains tools for raw data exploration: normalization, baselining, imputation of missing values, a powerful wrapper for amplification curve smoothing and a function to detect the start and end of an amplification curve. The capabilities of the software are enhanced by the implementation of algorithms unavailable in R: , such as a 5-point stencil for derivative interpolation. Simulation tools, statistical tests, plots for data quality management, amplification efficiency/quantification cycle calculation, and datasets from qPCR and qIA experiments are part of the package. Core functionalities are integrated in GUIs (web-based and standalone shiny applications), thus streamlining analysis and report generation. http://cran.r-project.org/web/packages/chipPCR. Source code: https://github.com/michbur/chipPCR. stefan.roediger@b-tu.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

PubMed Central

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

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

PubMed

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.

A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

PubMed

Rampazzo, Rita de Cássia Pontello; Solcà, Manuela da Silva; Santos, Liliane Celestino Sales; Pereira, Lais de Novaes; Guedes, José Carlos Oliveira; Veras, Patrícia Sampaio Tavares; Fraga, Deborah Bittencourt Mothé; Krieger, Marco Aurélio; Costa, Alexandre Dias Tavares

2017-11-15

Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format

Historical Cost Curves for Hydrogen Masers and Cesium Beam Frequency and Timing Standards

NASA Technical Reports Server (NTRS)

Remer, D. S.; Moore, R. C.

1985-01-01

Historical cost curves were developed for hydrogen masers and cesium beam standards used for frequency and timing calibration in the Deep Space Network. These curves may be used to calculate the cost of future hydrogen masers or cesium beam standards in either future or current dollars. The cesium beam standards are decreasing in cost by about 2.3% per year since 1966, and hydrogen masers are decreasing by about 0.8% per year since 1978 relative to the National Aeronautics and Space Administration inflation index.

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

PubMed Central

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 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 variation in qPCR experiments

How to Combine ChIP with qPCR.

PubMed

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.

Qualis-SIS: automated standard curve generation and quality assessment for multiplexed targeted quantitative proteomic experiments with labeled standards.

PubMed

Mohammed, Yassene; Percy, Andrew J; Chambers, Andrew G; Borchers, Christoph H

2015-02-06

Multiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.

Research on Standard and Automatic Judgment of Press-fit Curve of Locomotive Wheel-set Based on AAR Standard

NASA Astrophysics Data System (ADS)

Lu, Jun; Xiao, Jun; Gao, Dong Jun; Zong, Shu Yu; Li, Zhu

2018-03-01

In the production of the Association of American Railroads (AAR) locomotive wheel-set, the press-fit curve is the most important basis for the reliability of wheel-set assembly. In the past, Most of production enterprises mainly use artificial detection methods to determine the quality of assembly. There are cases of miscarriage of justice appear. For this reason, the research on the standard is carried out. And the automatic judgment of press-fit curve is analysed and designed, so as to provide guidance for the locomotive wheel-set production based on AAR standard.

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

USGS Publications Warehouse

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.

Relationship and Variation of qPCR and Culturable Enterococci Estimates in Ambient Surface Waters Are Predictable

EPA Science Inventory

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 meth...

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

PubMed Central

Mellerup, Anders; Ståhl, Marie

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. PMID:26114765

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

USGS Publications Warehouse

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.

Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

PubMed Central

Ackermann, Mark R.

2006-01-01

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition and to introduce/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems this invention solves are universal to all qPCR reactions, and it performs all necessary qPCR set-up calculations in about 52 seconds (using a pentium 4 processor) for up to seven qPCR targets and seventy-two samples at a time – calculations that commonly take capable investigators days to finish. We have named this custom Excel-based file system "FocusField2-6GallupqPCRSet-upTool-001" (FF2-6-001 qPCR set-up tool), and are in the process of transforming it into professional qPCR set-up software to be made available in 2007. The current prototype is already fully functional. PMID:17033699

Calibration-free assays on standard real-time PCR devices

PubMed Central

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-01-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration. PMID:28327545

Calibration-free assays on standard real-time PCR devices

NASA Astrophysics Data System (ADS)

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-03-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

PubMed Central

Rutledge, Robert G.

2011-01-01

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. PMID:21407812

Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

PubMed

Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

2018-02-01

Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

Estimating site index of ponderosa pine in Northern California...standard curves, soil series, stem analysis

Treesearch

Robert F. Powers

1972-01-01

Four sets of standard site index curves based on statewide or regionwide averages were compared with data on natural growth from nine young stands of ponderosa pine in northern California. The curves tested were by Meyer; Dunning; Dunning and Reineke; and Arvanitis, Lindquist, and Palley. The effects of soils on height growth were also studied. Among the curves tested...

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

USGS Publications Warehouse

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

Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters

EPA Science Inventory

In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laborator...

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

PubMed

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.

Comparison of culture and qPCR methods in detection of mycobacteria from drinking waters.

PubMed

Räsänen, Noora H J; Rintala, Helena; Miettinen, Ilkka T; Torvinen, Eila

2013-04-01

Environmental mycobacteria are common bacteria in man-made water systems and may cause infections and hypersensitivity pneumonitis via exposure to water. We compared a generally used cultivation method and a quantitative polymerase chain reaction (qPCR) method to detect mycobacteria in 3 types of drinking waters: surface water, ozone-treated surface water, and groundwater. There was a correlation between the numbers of mycobacteria obtained by cultivation and qPCR methods, but the ratio of the counts obtained by the 2 methods varied among the types of water. The qPCR counts in the drinking waters produced from surface or groundwater were 5 to 34 times higher than culturable counts. In ozone-treated surface waters, both methods gave similar counts. The ozone-treated drinking waters had the highest concentration of assimilable organic carbon, which may explain the good culturability. In warm tap waters, qPCR gave 43 times higher counts than cultivation, but both qPCR counts and culturable counts were lower than those in the drinking waters collected from the same sites. The TaqMan qPCR method is a rapid and sensitive tool for total quantitation of mycobacteria in different types of clean waters. The raw water source and treatments affect both culturability and total numbers of mycobacteria in drinking waters.

Development of a Dry-Reagent-Based qPCR to Facilitate the Diagnosis of Mycobacterium ulcerans Infection in Endemic Countries

PubMed Central

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

2015-01-01

Background 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. Methodology/Principal Findings 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. Conclusions 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. PMID:25830546

A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

EPA Science Inventory

In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

PubMed

Dorn-In, Samart; Fahn, Carmen; Hölzel, Christina S; Wenz, Sebastian; Hartwig, Isabella; Schwaiger, Karin; Bauer, Johann

2014-10-01

Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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

PubMed

Krøjgaard, Louise H; Krogfelt, Karen A; Albrechtsen, Hans-Jørgen; Uldum, Søren A

2011-11-21

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 for risk assessment. In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*105GU/L while L. pneumophila were detected in a range from LOQ to 6.8*105 GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*106 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 poor (r2 = 0.31 for culture and Legionella spp. assay, r2 = 0.20 for culture and L. pneumophila assay). Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

Droplet Digital PCR for Minimal Residual Disease Detection in Mature Lymphoproliferative Disorders.

PubMed

Drandi, Daniela; Ferrero, Simone; Ladetto, Marco

2018-01-01

Minimal residual disease (MRD) detection has a powerful prognostic relevance for response evaluation and prediction of relapse in hematological malignancies. Real-time quantitative PCR (qPCR) has become the settled and standardized method for MRD assessment in lymphoid disorders. However, qPCR is a relative quantification approach, since it requires a reference standard curve. Droplet digital TM PCR (ddPCR TM ) allows a reliable absolute tumor burden quantification withdrawing the need for preparing, for each experiment, a tumor-specific standard curve. We have recently shown that ddPCR has a good concordance with qPCR and could be a feasible and reliable tool for MRD monitoring in mature lymphoproliferative disorders. In this chapter we describe the experimental workflow, from the detection of the clonal molecular marker to the MRD monitoring by ddPCR, in patients affected by multiple myeloma, mantle cell lymphoma and follicular lymphoma. However, standardization programs among different laboratories are needed in order to ensure the reliability and reproducibility of ddPCR-based MRD results.

Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

PubMed Central

Pacheco, Ana Beatriz F.; Guedes, Iame A.; Azevedo, Sandra M.F.O.

2016-01-01

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. PMID:27338471

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

PubMed

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.

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

EPA Science Inventory

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 ...

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids.

PubMed

Nance, Shelly L; Riederer, Michael; Zubkowski, Tyler; Trudel, Marc; Rhodes, Linda D

2010-09-02

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR.

PubMed

Wang, Weilan; Zijlstra, Ruurd T; Gänzle, Michael G

2017-05-15

Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 10 5 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r 2  = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of

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

PubMed

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.

Nonlinear method for including the mass uncertainty of standards and the system measurement errors in the fitting of calibration curves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickles, W.L.; McClure, J.W.; Howell, R.H.

1978-01-01

A sophisticated non-linear multiparameter fitting program has been used to produce a best fit calibration curve for the response of an x-ray fluorescence analyzer to uranium nitrate, freeze dried, 0.2% accurate, gravimetric standards. The program is based on unconstrained minimization subroutine, VA02A. The program considers the mass values of the gravimetric standards as parameters to be fit along with the normal calibration curve parameters. The fitting procedure weights with the system errors and the mass errors in a consistent way. The resulting best fit calibration curve parameters reflect the fact that the masses of the standard samples are measured quantitiesmore » with a known error. Error estimates for the calibration curve parameters can be obtined from the curvature of the Chi-Squared Matrix or from error relaxation techniques. It has been shown that non-dispersive x-ray fluorescence analysis of 0.1 to 1 mg freeze-dried UNO/sub 3/ can have an accuracy of 0.2% in 1000 sec.« less

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

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

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 different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for

How to Avoid a Learning Curve in Stapedotomy: A Standardized Surgical Technique.

PubMed

Kwok, Pingling; Gleich, Otto; Dalles, Katharina; Mayr, Elisabeth; Jacob, Peter; Strutz, Jürgen

2017-08-01

To evaluate, whether a learning curve for beginners in stapedotomy can be avoided by using a prosthesis with thermal memory-shape attachment in combination with a standardized laser-assisted surgical technique. Retrospective case review. Tertiary referral center. Fifty-eight ears were operated by three experienced surgeons and compared with a group of 12 cases operated by a beginner in stapedotomy. Stapedotomy. Difference of pure-tone audiometry thresholds measured before and after surgery. The average postoperative gain for air conduction in the frequencies below 2 kHz was 20 to 25 dB and decreased for the higher frequencies. Using the Mann-Whitney-U test for comparing mean gain between experienced and inexperienced surgeons showed no significant difference (p = 0.281 at 4 kHz and p > 0.7 for the other frequencies). A Spearman rank correlation of the postoperative gain for air- and bone-conduction thresholds was obtained at each test frequency for the first 12 patients consecutively treated with a thermal memory-shape attachment prosthesis by two experienced and one inexperienced surgeon. This analysis does not support the hypothesis of a "learning effect" that should be associated with an improved outcome for successively treated patients. It is possible to avoid a learning curve in stapes surgery by applying a thermal memory-shape prosthesis in a standardized laser-assisted surgical procedure.

Probabilistic PCA of censored data: accounting for uncertainties in the visualization of high-throughput single-cell qPCR data.

PubMed

Buettner, Florian; Moignard, Victoria; Göttgens, Berthold; Theis, Fabian J

2014-07-01

High-throughput single-cell quantitative real-time polymerase chain reaction (qPCR) is a promising technique allowing for new insights in complex cellular processes. However, the PCR reaction can be detected only up to a certain detection limit, whereas failed reactions could be due to low or absent expression, and the true expression level is unknown. Because this censoring can occur for high proportions of the data, it is one of the main challenges when dealing with single-cell qPCR data. Principal component analysis (PCA) is an important tool for visualizing the structure of high-dimensional data as well as for identifying subpopulations of cells. However, to date it is not clear how to perform a PCA of censored data. We present a probabilistic approach that accounts for the censoring and evaluate it for two typical datasets containing single-cell qPCR data. We use the Gaussian process latent variable model framework to account for censoring by introducing an appropriate noise model and allowing a different kernel for each dimension. We evaluate this new approach for two typical qPCR datasets (of mouse embryonic stem cells and blood stem/progenitor cells, respectively) by performing linear and non-linear probabilistic PCA. Taking the censoring into account results in a 2D representation of the data, which better reflects its known structure: in both datasets, our new approach results in a better separation of known cell types and is able to reveal subpopulations in one dataset that could not be resolved using standard PCA. The implementation was based on the existing Gaussian process latent variable model toolbox (https://github.com/SheffieldML/GPmat); extensions for noise models and kernels accounting for censoring are available at http://icb.helmholtz-muenchen.de/censgplvm. © The Author 2014. Published by Oxford University Press. All rights reserved.

Probabilistic PCA of censored data: accounting for uncertainties in the visualization of high-throughput single-cell qPCR data

PubMed Central

Buettner, Florian; Moignard, Victoria; Göttgens, Berthold; Theis, Fabian J.

2014-01-01

Motivation: High-throughput single-cell quantitative real-time polymerase chain reaction (qPCR) is a promising technique allowing for new insights in complex cellular processes. However, the PCR reaction can be detected only up to a certain detection limit, whereas failed reactions could be due to low or absent expression, and the true expression level is unknown. Because this censoring can occur for high proportions of the data, it is one of the main challenges when dealing with single-cell qPCR data. Principal component analysis (PCA) is an important tool for visualizing the structure of high-dimensional data as well as for identifying subpopulations of cells. However, to date it is not clear how to perform a PCA of censored data. We present a probabilistic approach that accounts for the censoring and evaluate it for two typical datasets containing single-cell qPCR data. Results: We use the Gaussian process latent variable model framework to account for censoring by introducing an appropriate noise model and allowing a different kernel for each dimension. We evaluate this new approach for two typical qPCR datasets (of mouse embryonic stem cells and blood stem/progenitor cells, respectively) by performing linear and non-linear probabilistic PCA. Taking the censoring into account results in a 2D representation of the data, which better reflects its known structure: in both datasets, our new approach results in a better separation of known cell types and is able to reveal subpopulations in one dataset that could not be resolved using standard PCA. Availability and implementation: The implementation was based on the existing Gaussian process latent variable model toolbox (https://github.com/SheffieldML/GPmat); extensions for noise models and kernels accounting for censoring are available at http://icb.helmholtz-muenchen.de/censgplvm. Contact: fbuettner.phys@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24618470

Growth curves and the international standard: How children's growth reflects challenging conditions in rural Timor-Leste.

PubMed

Spencer, Phoebe R; Sanders, Katherine A; Judge, Debra S

2018-02-01

Population-specific growth references are important in understanding local growth variation, especially in developing countries where child growth is poor and the need for effective health interventions is high. In this article, we use mixed longitudinal data to calculate the first growth curves for rural East Timorese children to identify where, during development, deviation from the international standards occurs. Over an eight-year period, 1,245 children from two ecologically distinct rural areas of Timor-Leste were measured a total of 4,904 times. We compared growth to the World Health Organization (WHO) standards using z-scores, and modeled height and weight velocity using the SuperImposition by Translation And Rotation (SITAR) method. Using the Generalized Additive Model for Location, Scale and Shape (GAMLSS) method, we created the first growth curves for rural Timorese children for height, weight and body mass index (BMI). Relative to the WHO standards, children show early-life growth faltering, and stunting throughout childhood and adolescence. The median height and weight for this population tracks below the WHO fifth centile. Males have poorer growth than females in both z-BMI (p = .001) and z-height-for-age (p = .018) and, unlike females, continue to grow into adulthood. This is the most comprehensive investigation to date of rural Timorese children's growth, and the growth curves created may potentially be used to identify future secular trends in growth as the country develops. We show significant deviation from the international standard that becomes most pronounced at adolescence, similar to the growth of other Asian populations. Males and females show different growth responses to challenging conditions in this population. © 2017 Wiley Periodicals, Inc.

Stepwise kinetic equilibrium models of quantitative polymerase chain reaction.

PubMed

Cobbs, Gary

2012-08-16

initial target concentration. Model 1 was found to be slightly more robust than model 2 giving better estimates of initial target concentration when estimation of parameters was done for qPCR curves with very different initial target concentration. Both models may be used to estimate the initial absolute concentration of target sequence when a standard curve is not available. It is argued that the kinetic approach to modeling and interpreting quantitative PCR data has the potential to give more precise estimates of the true initial target concentrations than other methods currently used for analysis of qPCR data. The two models presented here give a unified model of the qPCR process in that they explain the shape of the qPCR curve for a wide variety of initial target concentrations.

Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus.

PubMed

Glover, William A; Atienza, Ederlyn E; Nesbitt, Shannon; Kim, Woo J; Castor, Jared; Cook, Linda; Jerome, Keith R

2016-01-01

Quantitative DNA detection of cytomegalovirus (CMV) and BK virus (BKV) is critical in the management of transplant patients. Quantitative laboratory-developed procedures for CMV and BKV have been described in which much of the processing is automated, resulting in rapid, reproducible, and high-throughput testing of transplant patients. To increase the efficiency of such assays, the performance and stability of four commercial preassembled frozen fast qPCR master mixes (Roche FastStart Universal Probe Master Mix with Rox, Bio-Rad SsoFast Probes Supermix with Rox, Life Technologies TaqMan FastAdvanced Master Mix, and Life Technologies Fast Universal PCR Master Mix), in combination with in-house designed primers and probes, was evaluated using controls and standards from standard CMV and BK assays. A subsequent parallel evaluation using patient samples was performed comparing the performance of freshly prepared assay mixes versus aliquoted frozen master mixes made with two of the fast qPCR mixes (Life Technologies TaqMan FastAdvanced Master Mix, and Bio-Rad SsoFast Probes Supermix with Rox), chosen based on their performance and compatibility with existing PCR cycling conditions. The data demonstrate that the frozen master mixes retain excellent performance over a period of at least 10 weeks. During the parallel testing using clinical specimens, no difference in quantitative results was observed between the preassembled frozen master mixes and freshly prepared master mixes. Preassembled fast real-time qPCR frozen master mixes perform well and represent an additional strategy laboratories can implement to reduce assay preparation times, and to minimize technical errors and effort necessary to perform clinical PCR. © 2015 Wiley Periodicals, Inc.

Use of a non-linear method for including the mass uncertainty of gravimetric standards and system measurement errors in the fitting of calibration curves for XRFA freeze-dried UNO/sub 3/ standards

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickles, W.L.; McClure, J.W.; Howell, R.H.

1978-05-01

A sophisticated nonlinear multiparameter fitting program was used to produce a best fit calibration curve for the response of an x-ray fluorescence analyzer to uranium nitrate, freeze dried, 0.2% accurate, gravimetric standards. The program is based on unconstrained minimization subroutine, VA02A. The program considers the mass values of the gravimetric standards as parameters to be fit along with the normal calibration curve parameters. The fitting procedure weights with the system errors and the mass errors in a consistent way. The resulting best fit calibration curve parameters reflect the fact that the masses of the standard samples are measured quantities withmore » a known error. Error estimates for the calibration curve parameters can be obtained from the curvature of the ''Chi-Squared Matrix'' or from error relaxation techniques. It was shown that nondispersive XRFA of 0.1 to 1 mg freeze-dried UNO/sub 3/ can have an accuracy of 0.2% in 1000 s.« less

Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

PubMed

Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

2013-12-01

Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study

PubMed Central

Okuma, Kazu; Yamochi, Tadanori; Sato, Tomoo; Sasaki, Daisuke; Hasegawa, Hiroo; Umeki, Kazumi; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Naruse, Isao; Yamagishi, Makoto; Nakashima, Makoto; Momose, Haruka; Araki, Kumiko; Mizukami, Takuo; Mizusawa, Saeko; Okada, Yoshiaki; Ochiai, Masaki; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Nosaka, Kisato; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Satake, Masahiro; Okayama, Akihiko; Mochizuki, Manabu; Izumo, Shuji; Saito, Shigeru; Itabashi, Kazuo; Kamihira, Shimeru; Yamaguchi, Kazunari; Watanabe, Toshiki

2015-01-01

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory. PMID:26292315

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

PubMed

Kuramitsu, Madoka; Okuma, Kazu; Yamochi, Tadanori; Sato, Tomoo; Sasaki, Daisuke; Hasegawa, Hiroo; Umeki, Kazumi; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Naruse, Isao; Yamagishi, Makoto; Nakashima, Makoto; Momose, Haruka; Araki, Kumiko; Mizukami, Takuo; Mizusawa, Saeko; Okada, Yoshiaki; Ochiai, Masaki; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Nosaka, Kisato; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Satake, Masahiro; Okayama, Akihiko; Mochizuki, Manabu; Izumo, Shuji; Saito, Shigeru; Itabashi, Kazuo; Kamihira, Shimeru; Yamaguchi, Kazunari; Watanabe, Toshiki; Hamaguchi, Isao

2015-11-01

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

PubMed

Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

2017-09-19

Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be

Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

PubMed

Bojolly, Daline; Doyen, Périne; Le Fur, Bruno; Christaki, Urania; Verrez-Bagnis, Véronique; Grard, Thierry

2017-02-01

Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

Reference genes for normalization of qPCR assays in sugarcane plants under water deficit.

PubMed

de Andrade, Larissa Mara; Dos Santos Brito, Michael; Fávero Peixoto Junior, Rafael; Marchiori, Paulo Eduardo Ribeiro; Nóbile, Paula Macedo; Martins, Alexandre Palma Boer; Ribeiro, Rafael Vasconcelos; Creste, Silvana

2017-01-01

Sugarcane ( Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.

METHODS TO CLASSIFY ENVIRONMENTAL SAMPLES BASED ON MOLD ANALYSES BY QPCR

EPA Science Inventory

Quantitative PCR (QPCR) analysis of molds in indoor environmental samples produces highly accurate speciation and enumeration data. In a number of studies, eighty of the most common or potentially problematic indoor molds were identified and quantified in dust samples from homes...

Using the weighted area under the net benefit curve for decision curve analysis.

PubMed

Talluri, Rajesh; Shete, Sanjay

2016-07-18

Risk prediction models have been proposed for various diseases and are being improved as new predictors are identified. A major challenge is to determine whether the newly discovered predictors improve risk prediction. Decision curve analysis has been proposed as an alternative to the area under the curve and net reclassification index to evaluate the performance of prediction models in clinical scenarios. The decision curve computed using the net benefit can evaluate the predictive performance of risk models at a given or range of threshold probabilities. However, when the decision curves for 2 competing models cross in the range of interest, it is difficult to identify the best model as there is no readily available summary measure for evaluating the predictive performance. The key deterrent for using simple measures such as the area under the net benefit curve is the assumption that the threshold probabilities are uniformly distributed among patients. We propose a novel measure for performing decision curve analysis. The approach estimates the distribution of threshold probabilities without the need of additional data. Using the estimated distribution of threshold probabilities, the weighted area under the net benefit curve serves as the summary measure to compare risk prediction models in a range of interest. We compared 3 different approaches, the standard method, the area under the net benefit curve, and the weighted area under the net benefit curve. Type 1 error and power comparisons demonstrate that the weighted area under the net benefit curve has higher power compared to the other methods. Several simulation studies are presented to demonstrate the improvement in model comparison using the weighted area under the net benefit curve compared to the standard method. The proposed measure improves decision curve analysis by using the weighted area under the curve and thereby improves the power of the decision curve analysis to compare risk prediction models in

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

PubMed Central

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

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

PubMed

Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

2013-07-01

Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management. Copyright © 2013 Elsevier Inc. All rights reserved.

Detection and quantification limits of the EPA Enterococcus qPCR method

EPA Science Inventory

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...

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

PubMed Central

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

[Growth standardized values and curves based on weight, length/height and head circumference for Chinese children under 7 years of age].

PubMed

Li, Hui

2009-03-01

To construct the growth standardized data and curves based on weight, length/height, head circumference for Chinese children under 7 years of age. Random cluster sampling was used. The fourth national growth survey of children under 7 years in the nine cities (Beijing, Harbin, Xi'an, Shanghai, Nanjing, Wuhan, Fuzhou, Guangzhou and Kunming) of China was performed in 2005 and from this survey, data of 69 760 urban healthy boys and girls were used to set up the database for weight-for-age, height-for-age (length was measured for children under 3 years) and head circumference-for-age. Anthropometric data were ascribed to rigorous methods of data collection and standardized procedures across study sites. LMS method based on BOX-COX normal transformation and cubic splines smoothing technique was chosen for fitting the raw data according to study design and data features, and standardized values of any percentile and standard deviation were obtained by the special formulation of L, M and S parameters. Length-for-age and height-for-age standards were constructed by fitting the same model but the final curves reflected the 0.7 cm average difference between these two measurements. A set of systematic diagnostic tools was used to detect possible biases in estimated percentiles or standard deviation curves, including chi2 test, which was used for reference to evaluate to the goodness of fit. The 3rd, 10th, 25th, 50th, 75th, 90th, 97th smoothed percentiles and -3, -2, -1, 0, +1, +2, +3 SD values and curves of weight-for-age, length/height-for-age and head circumference-for-age for boys and girls aged 0-7 years were made out respectively. The Chinese child growth charts was slightly higher than the WHO child growth standards. The newly established growth charts represented the growth level of healthy and well-nourished Chinese children. The sample size was very large and national, the data were high-quality and the smoothing method was internationally accepted. The new Chinese

Leishmania infection: laboratory diagnosing in the absence of a "gold standard".

PubMed

Rodríguez-Cortés, Alhelí; Ojeda, Ana; Francino, Olga; López-Fuertes, Laura; Timón, Marcos; Alberola, Jordi

2010-02-01

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2.

Development and optimization of an efficient qPCR system for olive authentication in edible oils.

PubMed

Alonso-Rebollo, Alba; Ramos-Gómez, Sonia; Busto, María D; Ortega, Natividad

2017-10-01

The applicability of qPCR in olive-oil authentication depends on the DNA obtained from the oils and the amplification primers. Therefore, four olive-specific amplification systems based on the trnL gene were designed (A-, B-, C- and D-trnL systems). The qPCR conditions, primer concentration and annealing temperature, were optimized. The systems were tested for efficiency and sensitivity to select the most suitable for olive oil authentication. The selected system (D-trnL) demonstrated specificity toward olive in contrast to other oleaginous species (canola, soybean, sunflower, maize, peanut and coconut) and showed high sensitivity in a broad linear dynamic range (LOD and LOQ: 500ng - 0.0625pg). This qPCR system enabled detection, with high sensitivity and specificity, of olive DNA isolated from oils processed in different ways, establishing it as an efficient method for the authentication of olive oil regardless of its category. Copyright © 2017 Elsevier Ltd. All rights reserved.

The 1-loop effective potential for the Standard Model in curved spacetime

NASA Astrophysics Data System (ADS)

Markkanen, Tommi; Nurmi, Sami; Rajantie, Arttu; Stopyra, Stephen

2018-06-01

The renormalisation group improved Standard Model effective potential in an arbitrary curved spacetime is computed to one loop order in perturbation theory. The loop corrections are computed in the ultraviolet limit, which makes them independent of the choice of the vacuum state and allows the derivation of the complete set of β-functions. The potential depends on the spacetime curvature through the direct non-minimal Higgs-curvature coupling, curvature contributions to the loop diagrams, and through the curvature dependence of the renormalisation scale. Together, these lead to significant curvature dependence, which needs to be taken into account in cosmological applications, which is demonstrated with the example of vacuum stability in de Sitter space.

Factors affecting the relationship between quantitative polymerase chain reaction (qPCR) and culture-based enumeration of Enterococcus in environmental waters.

PubMed

Raith, M R; Ebentier, D L; Cao, Y; Griffith, J F; Weisberg, S B

2014-03-01

To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class (qPCR or culture) and (iii) between-class difference. We analysed 306 samples using two culture-based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both qPCR methods indicated beach posting while both culture methods did not. Measurement target difference (DNA vs growth) accounted for the majority of the qPCR-vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. This is the first study to quantify the frequency with which culture-vs-qPCR discrepancies can be attributed to target difference - vs - method variability. © 2013 The Society for Applied Microbiology.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

PubMed

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the

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

ERIC Educational Resources Information Center

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…

Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples.

PubMed

Holmøy, Ingrid H; Toft, Nils; Jørgensen, Hannah J; Mørk, Tormod; Sølverød, Liv; Nødtvedt, Ane

2018-06-01

Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR. The within-herd prevalence in the six herds, as estimated by the latent class models ranged from 7.7 to 50.8%. At the recommended cut-off (cycle threshold 37), the sensitivity of the qPCR was significantly higher at 95.3 (95% posterior probability interval [PPI] [84.2; 99.6]) than that of bacteriological culture at 58.2 (95% PPI [43.8; 74.4]). However, bacterial culture had a higher specificity of 99.7 (95% PPI [98.5; 100.0]) compared to the qPCR at 98.5 (95% PPI [94.6; 99.9]). The median estimated negative predictive values of qPCR was consistently higher than those of the BC at all estimated prevalences, and the superiority of the qPCR increased with increasing within-herd prevalence. The median positive predictive values of BC was in general higher than the estimates for the qPCR, however, at the highest prevalence the predictive ability of both tests were similar. Copyright © 2018 Elsevier B.V. All

Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

USGS Publications Warehouse

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.

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

PubMed Central

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

7 CFR 42.142 - Curve for obtaining Operating Characteristic (OC) curve information for skip lot sampling and...

Code of Federal Regulations, 2010 CFR

2010-01-01

... the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... CONDITION OF FOOD CONTAINERS Miscellaneous § 42.142 Curve for obtaining Operating Characteristic (OC) curve...

Development of a viability standard curve for microencapsulated probiotic bacteria using confocal microscopy and image analysis software.

PubMed

Moore, Sarah; Kailasapathy, Kasipathy; Phillips, Michael; Jones, Mark R

2015-07-01

Microencapsulation is proposed to protect probiotic strains from food processing procedures and to maintain probiotic viability. Little research has described the in situ viability of microencapsulated probiotics. This study successfully developed a real-time viability standard curve for microencapsulated bacteria using confocal microscopy, fluorescent dyes and image analysis software. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

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

PubMed Central

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

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. PMID:24626408

Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

PubMed

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

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.

Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

NASA Astrophysics Data System (ADS)

Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

2017-06-01

Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Waist Circumferences of Chilean Students: Comparison of the CDC-2012 Standard and Proposed Percentile Curves

PubMed Central

Gómez-Campos, Rossana; Lee Andruske, Cinthya; Hespanhol, Jefferson; Sulla Torres, Jose; Arruda, Miguel; Luarte-Rocha, Cristian; Cossio-Bolaños, Marco Antonio

2015-01-01

The measurement of waist circumference (WC) is considered to be an important means to control overweight and obesity in children and adolescents. The objectives of the study were to (a) compare the WC measurements of Chilean students with the international CDC-2012 standard and other international standards, and (b) propose a specific measurement value for the WC of Chilean students based on age and sex. A total of 3892 students (6 to 18 years old) were assessed. Weight, height, body mass index (BMI), and WC were measured. WC was compared with the CDC-2012 international standard. Percentiles were constructed based on the LMS method. Chilean males had a greater WC during infancy. Subsequently, in late adolescence, males showed values lower than those of the international standards. Chilean females demonstrated values similar to the standards until the age of 12. Subsequently, females showed lower values. The 85th and 95th percentiles were adopted as cutoff points for evaluating overweight and obesity based on age and sex. The WC of Chilean students differs from the CDC-2012 curves. The regional norms proposed are a means to identify children and adolescents with a high risk of suffering from overweight and obesity disorders. PMID:26184250

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

EPA Science Inventory

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 ...

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

PubMed

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

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...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

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...

Curve fitting air sample filter decay curves to estimate transuranic content.

PubMed

Hayes, Robert B; Chiou, Hung Cheng

2004-01-01

By testing industry standard techniques for radon progeny evaluation on air sample filters, a new technique is developed to evaluate transuranic activity on air filters by curve fitting the decay curves. The industry method modified here is simply the use of filter activity measurements at different times to estimate the air concentrations of radon progeny. The primary modification was to not look for specific radon progeny values but rather transuranic activity. By using a method that will provide reasonably conservative estimates of the transuranic activity present on a filter, some credit for the decay curve shape can then be taken. By carrying out rigorous statistical analysis of the curve fits to over 65 samples having no transuranic activity taken over a 10-mo period, an optimization of the fitting function and quality tests for this purpose was attained.

Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters.

PubMed

Cao, Y; Griffith, J F; Dorevitch, S; Weisberg, S B

2012-07-01

  Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false-negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition.   Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54-85% agreement between ICs and serial dilution.   The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable.   Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

DEVELOPMENT OF CRASSPHAGE-BASED QPCR ASSAYS ...

EPA Pesticide Factsheets

A newly discovered bacteriophage, “crAssphage”, is predicted to be both highlyabundant and predominantly human-associated, both ideal characteristics for a human-specific fecal indicator. A total of 384 end-point PCR primers were designed along the length of the crAssphage genome, eliminating regions suspected to be hypervariable or react with other animal sources. The primer pairs were rigorously tested in three rounds of screening for specificity, geographic variability, limit of detection, and environmental water performance. The two best performing assays, crAss056 and crAss064, were adapted to a qPCR platform and exhibited a specificity of 98.0% and 98.9%, respectively. The markers’ abundance was compared with two bacterial based assays and were found at concentrations at or above the bacterial based assays in wastewater influent and impacted environmental waters. This poster will present the methodology of the novel marker development and the potential uses for this technology in maintaining sustainable waterways in the future. To inform the public.

QPCR Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches

EPA Science Inventory

The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the ...

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

PubMed Central

Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

2011-01-01

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

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

PubMed

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.

Quantification by qPCR of Pathobionts in Chronic Periodontitis: Development of Predictive Models of Disease Severity at Site-Specific Level.

PubMed

Tomás, Inmaculada; Regueira-Iglesias, Alba; López, Maria; Arias-Bujanda, Nora; Novoa, Lourdes; Balsa-Castro, Carlos; Tomás, Maria

2017-01-01

Currently, there is little evidence available on the development of predictive models for the diagnosis or prognosis of chronic periodontitis based on the qPCR quantification of subgingival pathobionts. Our objectives were to: (1) analyze and internally validate pathobiont-based models that could be used to distinguish different periodontal conditions at site-specific level within the same patient with chronic periodontitis; (2) develop nomograms derived from predictive models. Subgingival plaque samples were obtained from control and periodontal sites (probing pocket depth and clinical attachment loss <4 mm and >4 mm, respectively) from 40 patients with moderate-severe generalized chronic periodontitis. The samples were analyzed by qPCR using TaqMan probes and specific primers to determine the concentrations of Actinobacillus actinomycetemcomitans (Aa) , Fusobacterium nucleatum (Fn) , Parvimonas micra (Pm) , Porphyromonas gingivalis (Pg) , Prevotella intermedia (Pi) , Tannerella forsythia (Tf) , and Treponema denticola (Td) . The pathobiont-based models were obtained using multivariate binary logistic regression. The best models were selected according to specified criteria. The discrimination was assessed using receiver operating characteristic curves and numerous classification measures were thus obtained. The nomograms were built based on the best predictive models. Eight bacterial cluster-based models showed an area under the curve (AUC) ≥0.760 and a sensitivity and specificity ≥75.0%. The PiTfFn cluster showed an AUC of 0.773 (sensitivity and specificity = 75.0%). When Pm and AaPm were incorporated in the TdPiTfFn cluster, we detected the two best predictive models with an AUC of 0.788 and 0.789, respectively (sensitivity and specificity = 77.5%). The TdPiTfAa cluster had an AUC of 0.785 (sensitivity and specificity = 75.0%). When Pm was incorporated in this cluster, a new predictive model appeared with better AUC and specificity values (0.787 and 80

Quantification by qPCR of Pathobionts in Chronic Periodontitis: Development of Predictive Models of Disease Severity at Site-Specific Level

PubMed Central

Tomás, Inmaculada; Regueira-Iglesias, Alba; López, Maria; Arias-Bujanda, Nora; Novoa, Lourdes; Balsa-Castro, Carlos; Tomás, Maria

2017-01-01

Currently, there is little evidence available on the development of predictive models for the diagnosis or prognosis of chronic periodontitis based on the qPCR quantification of subgingival pathobionts. Our objectives were to: (1) analyze and internally validate pathobiont-based models that could be used to distinguish different periodontal conditions at site-specific level within the same patient with chronic periodontitis; (2) develop nomograms derived from predictive models. Subgingival plaque samples were obtained from control and periodontal sites (probing pocket depth and clinical attachment loss <4 mm and >4 mm, respectively) from 40 patients with moderate-severe generalized chronic periodontitis. The samples were analyzed by qPCR using TaqMan probes and specific primers to determine the concentrations of Actinobacillus actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Parvimonas micra (Pm), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and Treponema denticola (Td). The pathobiont-based models were obtained using multivariate binary logistic regression. The best models were selected according to specified criteria. The discrimination was assessed using receiver operating characteristic curves and numerous classification measures were thus obtained. The nomograms were built based on the best predictive models. Eight bacterial cluster-based models showed an area under the curve (AUC) ≥0.760 and a sensitivity and specificity ≥75.0%. The PiTfFn cluster showed an AUC of 0.773 (sensitivity and specificity = 75.0%). When Pm and AaPm were incorporated in the TdPiTfFn cluster, we detected the two best predictive models with an AUC of 0.788 and 0.789, respectively (sensitivity and specificity = 77.5%). The TdPiTfAa cluster had an AUC of 0.785 (sensitivity and specificity = 75.0%). When Pm was incorporated in this cluster, a new predictive model appeared with better AUC and specificity values (0.787 and 80

Effect of Environmental Parameters on the qPCR Signal of Enterococci in Tropical Waters

EPA Science Inventory

Fecal contamination is the major source of pathogens in recreational waters. The need for quick public notifications has expanded the interest in the use of a rapid, quantitative polymerase chain reaction method (qPCR) to determine enterococci density. However, very little info...

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow-FISH techniques.

PubMed

Wand, Taylor; Fang, Mike; Chen, Christina; Hardy, Nathan; McCoy, J Philip; Dumitriu, Bogdan; Young, Neal S; Biancotto, Angélique

2016-10-01

Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA) 3 probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters.

PubMed

Converse, Reagan R; Blackwood, A Denene; Kirs, Marek; Griffith, John F; Noble, Rachel T

2009-11-01

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.

The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs.

PubMed

Garcia, João Luis; Burrells, Alison; Bartley, Paul M; Bartley, Kathryn; Innes, Elisabeth A; Katzer, Frank

2017-12-01

In the present study we experimentally infected pigs with T. gondii tachyzoites, bradyzoites and oocysts in order to evaluate IgG-ELISA, nested-PCR, and qPCR for diagnosis of ocular infection. Eighteen pigs were divided into four groups: G1 (infected with 10 3 tissue cysts of the M4 strain (type II) at day 28, n=5), G2 (infected with 10 3 oocysts of the M4 strain at day 28, n=5), G3 (infected with tachyzoites of S48 strain (type 1) at day 0, n=5), and G4 (uninfected unchallenged, control group n=3). At day 70 of the experiment all animals were culled, and serum, aqueous humor (AH) and vitreous humor (VH) samples were collected to perform indirect ELISA, and PCR (nPCR, and qPCR). By ELISA nine pigs (60%) out of 15 were positive in VH samples, and seven out of 15 (46%) were positive in AH samples. Both molecular techniques used here, nPCR and qPCR, were able to detect <50fg of T. gondii tachyzoite DNA. The nPCR and qPCR detected six (7/15, 47%) and two (2/15, 13.3%) positive animals respectively. Antibody responses were detected in serum and in AH and VH from the eye, suggesting that pigs may be an animal that could be used as a model to further our understanding of diagnosis of human ocular infection with T. gondii. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adapting Shape Parameters for Cubic Bezier Curves

NASA Technical Reports Server (NTRS)

Isacoff, D.; Bailey, M. J.

1985-01-01

Bezier curves are an established tool in Computer Aided Geometric Design. One of the drawbacks of the Bezier method is that the curves often bear little resemblance to their control polygons. As a result, it becomes increasingly difficult to obtain anything but a rough outline of the desired shape. One possible solution is tomanipulate the curve itself instead of the control polygon. The standard cubic Bezier curve form has introduced into it two shape parameters, gamma 1 and 2. These parameters give the user the ability to manipulate the curve while the control polygon retains its original form, thereby providing a more intuitive feel for the necessary changes to the curve in order to achieve the desired shape.

Development and Use of Fluorescent Antibody and qPCR Protocols for the Electrostatic Spore Trap

USDA-ARS?s Scientific Manuscript database

Fluorescent antibody (FA) and qPCR protocols were evaluated for the newly developed aerobiological sampler (Ionic Spore Trap), which depends upon electrostatic deposition of particulates onto a 25 mm aluminum disk (stub). This device was originally designed for assessment of captured particulates by...

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

A multiplex real-time PCR assay, based on invA and pagC genes, for the detection and quantification of Salmonella enterica from cattle lymph nodes.

PubMed

Bai, Jianfa; Trinetta, Valentina; Shi, Xiaorong; Noll, Lance W; Magossi, Gabriela; Zheng, Wanglong; Porter, Elizabeth P; Cernicchiaro, Natalia; Renter, David G; Nagaraja, Tiruvoor G

2018-05-01

Cattle lymph nodes can harbor Salmonella and potentially contaminate beef products. We have developed and validated a new real-time PCR (qPCR) assay for the detection and quantification of Salmonella enterica in cattle lymph nodes. The assay targets both the invA and pagC genes, the most conserved molecular targets in Salmonella enterica. An 18S rRNA gene assay that amplifies from cattle and other animal species was also included as an internal control. Available DNA sequences for invA, pagC and 18S rRNA genes were used for primer and probe selections. Three Salmonella serotypes, S. Typhimurium, S. Anatum, and S. Montevideo, were used to assess the assay's analytical sensitivity. Correlation coefficients of standard curves generated for each target and for all three serotypes were >99% and qPCR amplification efficiencies were between 93% and 110%. Assay sensitivity was also determined using standard curve data generated from Salmonella-negative cattle lymph nodes spiked with 10-fold dilutions of the three Salmonella serotypes. Assay specificity was determined using Salmonella culture method, and qPCR testing on 36 Salmonella strains representing 33 serotypes, 38 Salmonella strains of unknown serotypes, 252 E. coli strains representing 40 serogroups, and 31 other bacterial strains representing 18 different species. A collection of 647 cattle lymph node samples from steers procured from the Midwest region of the US were tested by the qPCR, and compared to culture-method of detection. Salmonella prevalence by qPCR for pre-enriched and enriched lymph nodes was 19.8% (128/647) and 94.9% (614/647), respectively. A majority of qPCR positive pre-enriched samples (105/128) were at concentrations between 10 4 and 10 5  CFU/mL. Culture method detected Salmonella in 7.7% (50/647) and 80.7% (522/647) of pre- and post-enriched samples, respectively; 96.0% (48/50) of pre-enriched and 99.4% (519/522) of post-enriched culture-positive samples were also positive by qPCR. More

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.

PubMed

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.

Improving qPCR methodology for detection of foaming bacteria by analysis of broad-spectrum primers and a highly specific probe for quantification of Nocardia spp. in activated sludge.

PubMed

Asvapathanagul, P; Olson, B H

2017-01-01

To develop qPCR broad-spectrum primers combined with a Nocardia genus-specific probe for the identification of a broad spectrum of Nocardia spp. and to analyse the effects of using this developed primer and probe set on the ability to quantify Nocardia spp. in mixed DNA. The consequences of using a degenerative primer set and species-specific probe for the genus Nocardia on qPCR assays were examined using DNA extracts of pure cultures and activated sludge. The mixed DNA extracts where the target organism Nocardia flavorosea concentration ranged from 5 × 10 2 to 5 × 10 6 copies per reaction, while the background organism's DNA (Mycobacterium bovis) concentration was held at 5 × 10 6 copies per reaction, only produced comparable cycle threshold florescence levels when N. flavorosea concentration was greater than or equal to the background organism concentration. When concentrations of N. flavorosea were lowered in increments of 1 log, while holding M. bovis concentrations constant at 5 × 10 6 copies per reaction, all assays demonstrated delayed cycle threshold values with a maximum 34·6-fold decrease in cycle threshold at a ratio of 10 6 M. bovis: 10 2 N. flavorosea copies per reaction. The data presented in this study indicated that increasing the ability of a primer set to capture a broad group of organisms can affect the accuracy of quantification even when a highly specific probe is used. This study examined several applications of molecular tools in complex communities such as evaluating the effect of mispriming vs interference. It also elucidates the importance of understanding the community genetic make-up on primer design. Degenerative primers are very useful in amplifying bacterial DNA across genera, but reduce the efficiency of qPCR reactions. Therefore, standards that address closely related background species must be used to obtain accurate qPCR results. © 2016 The Society for Applied Microbiology.

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

2013-02-14

Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

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

PubMed

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.

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

PubMed

Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER

EPA Science Inventory

Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly...

Environmental standards for ionizing radiation: theoretical basis for dose-response curves.

PubMed Central

Upton, A C

1983-01-01

The types of injury attributable to ionizing radiation are subdivided, for purposes of risk assessment and radiological protection, into two broad categories: stochastic effects and nonstochastic effects. Stochastic effects are viewed as probablistic phenomena, varying in frequency but not severity as a function of the dose, without any threshold; nonstochastic effects are viewed as deterministic phenomena, varying in both frequency and severity as a function of the dose, with clinical thresholds. Included among stochastic effects are heritable effects (mutations and chromosome aberrations) and carcinogenic effects. Both types of effects are envisioned as unicellular phenomena which can result from nonlethal injury of individual cells, without the necessity of damage to other cells. For the induction of mutations and chromosome aberrations in the low-to-intermediate dose range, the dose-response curve with high-linear energy transfer (LET) radiation generally conforms to a linear nonthreshold relationship and varies relatively little with the dose rate. In contrast, the curve with low-LET radiation generally conforms to a linear-quadratic relationship, rising less steeply than the curve with high-LET radiation and increasing in slope with increasing dose and dose rate. The dose-response curve for carcinogenic effects varies widely from one type of neoplasm to another in the intermediate-to-high dose range, in part because of differences in the way large doses of radiation can affect the promotion and progression of different neoplasms. Information about dose-response relations for low-level irradiation is fragmentary but consistent, in general, with the hypothesis that the neoplastic transformation may result from mutation, chromosome aberration or genetic recombination in a single susceptible cell. PMID:6653536

Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium.

PubMed

Tesena, Parichart; Korchunjit, Wasamon; Taylor, Jane; Wongtawan, Tuempong

2017-01-01

Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol ™ , GENEzol ™ and MiniPrep ™ ) and four qPCR master mixes with SYBR ® green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.

Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

PubMed

Wang, Li-Ping; Lei, Kun

2016-12-01

Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Curve numbers for no-till: field data versus standard tables

USDA-ARS?s Scientific Manuscript database

The Curve Number procedure developed by Soil Conservation Service (Now Natural Resources Conservation Service) in the mid-1950s for estimating direct runoff from rainstorms has not been extensively tested in cropping systems under no-till. Analysis of CNs using the frequency matching and asymptotic ...

Standardized Percentile Curves of Body Mass Index of Northeast Iranian Children Aged 25 to 60 Months

PubMed Central

Emdadi, Maryam; Safarian, Mohammad; Doosti, Hassan

2011-01-01

Objective Growth charts are widely used to assess children's growth status and can provide a trajectory of growth during early important months of life. Racial differences necessitate using local growth charts. This study aimed to provide standardized growth curves of body mass index (BMI) for children living in northeast Iran. Methods A total of 23730 apparently healthy boys and girls aged 25 to 60 months recruited for 20 days from those attending community clinics for routine health checks. Anthropometric measurements were done by trained health staff using WHO methodology. The LMSP method with maximum penalized likelihood, the Generalized Additive Models, the Box-Cox power exponential distribution distribution, Akaike Information Criteria and Generalized Akaike Criteria with penalty equal to 3 [GAIC(3)], and Worm plot and Q-tests as goodness of fit tests were used to construct the centile reference charts. Findings The BMI centile curves for boys and girls aged 25 to 60 months were drawn utilizing a population of children living in northeast Iran. Conclusion The results of the current study demonstrate the possibility of preparation of local growth charts and their importance in evaluating children's growth. Also their differences, relative to those prepared by global references, reflect the necessity of preparing local charts in future studies using longitudinal data. PMID:23056770

Interaction Analysis of Longevity Interventions Using Survival Curves.

PubMed

Nowak, Stefan; Neidhart, Johannes; Szendro, Ivan G; Rzezonka, Jonas; Marathe, Rahul; Krug, Joachim

2018-01-06

A long-standing problem in ageing research is to understand how different factors contributing to longevity should be expected to act in combination under the assumption that they are independent. Standard interaction analysis compares the extension of mean lifespan achieved by a combination of interventions to the prediction under an additive or multiplicative null model, but neither model is fundamentally justified. Moreover, the target of longevity interventions is not mean life span but the entire survival curve. Here we formulate a mathematical approach for predicting the survival curve resulting from a combination of two independent interventions based on the survival curves of the individual treatments, and quantify interaction between interventions as the deviation from this prediction. We test the method on a published data set comprising survival curves for all combinations of four different longevity interventions in Caenorhabditis elegans . We find that interactions are generally weak even when the standard analysis indicates otherwise.

Interaction Analysis of Longevity Interventions Using Survival Curves

PubMed Central

Nowak, Stefan; Neidhart, Johannes; Szendro, Ivan G.; Rzezonka, Jonas; Marathe, Rahul; Krug, Joachim

2018-01-01

A long-standing problem in ageing research is to understand how different factors contributing to longevity should be expected to act in combination under the assumption that they are independent. Standard interaction analysis compares the extension of mean lifespan achieved by a combination of interventions to the prediction under an additive or multiplicative null model, but neither model is fundamentally justified. Moreover, the target of longevity interventions is not mean life span but the entire survival curve. Here we formulate a mathematical approach for predicting the survival curve resulting from a combination of two independent interventions based on the survival curves of the individual treatments, and quantify interaction between interventions as the deviation from this prediction. We test the method on a published data set comprising survival curves for all combinations of four different longevity interventions in Caenorhabditis elegans. We find that interactions are generally weak even when the standard analysis indicates otherwise. PMID:29316622

Solid-state curved focal plane arrays

NASA Technical Reports Server (NTRS)

Jones, Todd (Inventor); Nikzad, Shouleh (Inventor); Hoenk, Michael (Inventor)

2010-01-01

The present invention relates to curved focal plane arrays. More specifically, the present invention relates to a system and method for making solid-state curved focal plane arrays from standard and high-purity devices that may be matched to a given optical system. There are two ways to make a curved focal plane arrays starting with the fully fabricated device. One way, is to thin the device and conform it to a curvature. A second way, is to back-illuminate a thick device without making a thinned membrane. The thick device is a special class of devices; for example devices fabricated with high purity silicon. One surface of the device (the non VLSI fabricated surface, also referred to as the back surface) can be polished to form a curved surface.

Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

NASA Astrophysics Data System (ADS)

Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

2015-01-01

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

DOE PAGES

Stulberg, Michael J.; Huang, Qi

2015-10-01

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 regionsmore » 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. Moreover, 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.« less

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

PubMed Central

Stulberg, Michael J.; Huang, Qi

2015-01-01

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. PMID:26426354

Characterization of vaginal Lactobacillus species by rplK -based multiplex qPCR in Russian women.

PubMed

Demkin, Vladimir V; Koshechkin, Stanislav I

2017-10-01

We describe a multiplex qPCR assay for identification and quantitative assessment of a set of vaginal Lactobacillus species, including L. acidophilus, L. crispatus, L. gasseri, L. helveticus, L. iners, and L. jensenii. The assay extends the previously developed qPCR method for Lactobacillus detection and total quantification based on targeting the rplK gene. Both assays use only single pair of primers and a set of probes combined in three reactions, comprising a vaginal Lactobacillus diagnostic assay panel. The utility of the diagnostic panel was evaluated by analyzing of vaginal swab specimens from 145 patients with different status of vaginal health. Most frequently, only one Lactobacillus species was dominant (68,9%), mostly L. crispatus (18,6%) or L. iners (33,1%), but two or three Lactobacillus species were also being simultaneously detected (24,9%). The diagnostic panel will facilitate investigations of the role of Lactobacillus species in the health of the female reproductive system and promote studies of variability of the vaginal microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

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

EPA Science Inventory

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...

49 CFR 213.59 - Elevation of curved track; runoff.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 4 2011-10-01 2011-10-01 false Elevation of curved track; runoff. 213.59 Section 213.59 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION TRACK SAFETY STANDARDS Track Geometry § 213.59 Elevation of curved...

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

EPA Science Inventory

There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

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

USDA-ARS?s Scientific Manuscript database

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...

Estimating the Area Under ROC Curve When the Fitted Binormal Curves Demonstrate Improper Shape.

PubMed

Bandos, Andriy I; Guo, Ben; Gur, David

2017-02-01

The "binormal" model is the most frequently used tool for parametric receiver operating characteristic (ROC) analysis. The binormal ROC curves can have "improper" (non-concave) shapes that are unrealistic in many practical applications, and several tools (eg, PROPROC) have been developed to address this problem. However, due to the general robustness of binormal ROCs, the improperness of the fitted curves might carry little consequence for inferences about global summary indices, such as the area under the ROC curve (AUC). In this work, we investigate the effect of severe improperness of fitted binormal ROC curves on the reliability of AUC estimates when the data arise from an actually proper curve. We designed theoretically proper ROC scenarios that induce severely improper shape of fitted binormal curves in the presence of well-distributed empirical ROC points. The binormal curves were fitted using maximum likelihood approach. Using simulations, we estimated the frequency of severely improper fitted curves, bias of the estimated AUC, and coverage of 95% confidence intervals (CIs). In Appendix S1, we provide additional information on percentiles of the distribution of AUC estimates and bias when estimating partial AUCs. We also compared the results to a reference standard provided by empirical estimates obtained from continuous data. We observed up to 96% of severely improper curves depending on the scenario in question. The bias in the binormal AUC estimates was very small and the coverage of the CIs was close to nominal, whereas the estimates of partial AUC were biased upward in the high specificity range and downward in the low specificity range. Compared to a non-parametric approach, the binormal model led to slightly more variable AUC estimates, but at the same time to CIs with more appropriate coverage. The improper shape of the fitted binormal curve, by itself, ie, in the presence of a sufficient number of well-distributed points, does not imply

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

PubMed

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

2017-10-05

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. Copyright © 2017 Falabella et al.

Real-time PCR to supplement gold-standard culture-based detection of Legionella in environmental samples.

PubMed

Collins, S; Jorgensen, F; Willis, C; Walker, J

2015-10-01

Culture remains the gold-standard for the enumeration of environmental Legionella. However, it has several drawbacks including long incubation and poor sensitivity, causing delays in response times to outbreaks of Legionnaires' disease. This study aimed to validate real-time PCR assays to quantify Legionella species (ssrA gene), Legionella pneumophila (mip gene) and Leg. pneumophila serogroup-1 (wzm gene) to support culture-based detection in a frontline public health laboratory. Each qPCR assay had 100% specificity, excellent sensitivity (5 GU/reaction) and reproducibility. Comparison of the assays to culture-based enumeration of Legionella from 200 environmental samples showed that they had a negative predictive value of 100%. Thirty eight samples were positive for Legionella species by culture and qPCR. One hundred samples were negative by both methods, whereas 62 samples were negative by culture but positive by qPCR. The average log10 increase between culture and qPCR for Legionella spp. and Leg. pneumophila was 0·72 (P = 0·0002) and 0·51 (P = 0·006), respectively. The qPCR assays can be conducted on the same 1 l water sample as culture thus can be used as a supplementary technique to screen out negative samples and allow more rapid indication of positive samples. The assay could prove informative in public health investigations to identify or rule out sources of Legionella as well as to specifically identify Leg. pneumophila serogroup 1 in a timely manner not possible with culture. © 2015 The Society for Applied Microbiology.

Comparison of salivary collection and processing methods for quantitative HHV-8 detection.

PubMed

Speicher, D J; Johnson, N W

2014-10-01

Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown. Stabilising fluids impede nucleic acid degradation, compared with collection onto ice and then freezing, and we have shown that the DNA Genotek P-021 prototype kit (P-021) can produce high-quality DNA after 14 months of storage at room temperature. Here we evaluate the quantitative capability of 10 collection/processing methods. Unstimulated whole mouth fluid was spiked with a mixture of HHV-8 cloned constructs, 10-fold serial dilutions were produced, and samples were extracted and then examined with quantitative PCR (qPCR). Calibration curves were compared by linear regression and qPCR dynamics. All methods extracted with commercial spin columns produced linear calibration curves with large dynamic range and gave accurate viral loads. Ethanol precipitation of the P-021 does not produce a linear standard curve, and virus is lost in the cell pellet. DNA extractions from the P-021 using commercial spin columns produced linear standard curves with wide dynamic range and excellent limit of detection. When extracted with spin columns, the P-021 enables accurate viral loads down to 23 copies μl(-1) DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

PubMed Central

Kang, Shih-Ting; Hsieh, Yi-Shan; Feng, Chi-Ting; Chen, Yu-Ting; Yang, Pok Eric; Chen, Wei-Ming

2018-01-01

MicroRNAs (miRNAs) are 18–25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs’ short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences’ platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers. PMID:29208706

Molecular Quantification of Zooplankton Gut Content: The Case For qPCR

NASA Astrophysics Data System (ADS)

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.

Laparoscopic colorectal surgery in learning curve: Role of implementation of a standardized technique and recovery protocol. A cohort study

PubMed Central

Luglio, Gaetano; De Palma, Giovanni Domenico; Tarquini, Rachele; Giglio, Mariano Cesare; Sollazzo, Viviana; Esposito, Emanuela; Spadarella, Emanuela; Peltrini, Roberto; Liccardo, Filomena; Bucci, Luigi

2015-01-01

Background Despite the proven benefits, laparoscopic colorectal surgery is still under utilized among surgeons. A steep learning is one of the causes of its limited adoption. Aim of the study is to determine the feasibility and morbidity rate after laparoscopic colorectal surgery in a single institution, “learning curve” experience, implementing a well standardized operative technique and recovery protocol. Methods The first 50 patients treated laparoscopically were included. All the procedures were performed by a trainee surgeon, supervised by a consultant surgeon, according to the principle of complete mesocolic excision with central vascular ligation or TME. Patients underwent a fast track recovery programme. Recovery parameters, short-term outcomes, morbidity and mortality have been assessed. Results Type of resections: 20 left side resections, 8 right side resections, 14 low anterior resection/TME, 5 total colectomy and IRA, 3 total panproctocolectomy and pouch. Mean operative time: 227 min; mean number of lymph-nodes: 18.7. Conversion rate: 8%. Mean time to flatus: 1.3 days; Mean time to solid stool: 2.3 days. Mean length of hospital stay: 7.2 days. Overall morbidity: 24%; major morbidity (Dindo–Clavien III): 4%. No anastomotic leak, no mortality, no 30-days readmission. Conclusion Proper laparoscopic colorectal surgery is safe and leads to excellent results in terms of recovery and short term outcomes, even in a learning curve setting. Key factors for better outcomes and shortening the learning curve seem to be the adoption of a standardized technique and training model along with the strict supervision of an expert colorectal surgeon. PMID:25859386

Complexity of Curved Glass Structures

NASA Astrophysics Data System (ADS)

Kosić, T.; Svetel, I.; Cekić, Z.

2017-11-01

Despite the increasing number of research on the architectural structures of curvilinear forms and technological and practical improvement of the glass production observed over recent years, there is still a lack of comprehensive codes and standards, recommendations and experience data linked to real-life curved glass structures applications regarding design, manufacture, use, performance and economy. However, more and more complex buildings and structures with the large areas of glass envelope geometrically complex shape are built every year. The aim of the presented research is to collect data on the existing design philosophy on curved glass structure cases. The investigation includes a survey about how architects and engineers deal with different design aspects of curved glass structures with a special focus on the design and construction process, glass types and structural and fixing systems. The current paper gives a brief overview of the survey findings.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

PubMed

Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

2015-10-01

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.

Rapid quantification of plant-powdery mildew interactions by qPCR and conidiospore counts.

PubMed

Weßling, Ralf; Panstruga, Ralph

2012-08-31

The powdery mildew disease represents a valuable patho-system to study the interaction between plant hosts and obligate biotrophic fungal pathogens. Numerous discoveries have been made on the basis of the quantitative evaluation of plant-powdery mildew interactions, especially in the context of hyper-susceptible and/or resistant plant mutants. However, the presently available methods to score the pathogenic success of powdery mildew fungi are laborious and thus not well suited for medium- to high-throughput analysis. Here we present two new protocols that allow the rapid quantitative assessment of powdery mildew disease development. One procedure depends on quantitative polymerase chain reaction (qPCR)-based evaluation of fungal biomass, while the other relies on the quantification of fungal conidiospores. We validated both techniques using the powdery mildew pathogen Golovinomyces orontii on a set of hyper-susceptible and resistant Arabidopsis thaliana mutants and found that both cover a wide dynamic range of one to two (qPCR) and four to five (quantification of conidia) orders of magnitude, respectively. The two approaches yield reproducible results and are easy to perform without specialized equipment. The qPCR and spore count assays rapidly and reproducibly quantify powdery mildew pathogenesis. Our methods are performed at later stages of infection and discern mutant phenotypes accurately. The assays therefore complement currently used procedures of powdery mildew quantification and can overcome some of their limitations. In addition, they can easily be adapted to other plant-powdery mildew patho-systems.

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

EPA Science Inventory

EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM a...

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

PubMed Central

Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

2014-01-01

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

Principal Curves on Riemannian Manifolds.

PubMed

Hauberg, Soren

2016-09-01

Euclidean statistics are often generalized to Riemannian manifolds by replacing straight-line interpolations with geodesic ones. While these Riemannian models are familiar-looking, they are restricted by the inflexibility of geodesics, and they rely on constructions which are optimal only in Euclidean domains. We consider extensions of Principal Component Analysis (PCA) to Riemannian manifolds. Classic Riemannian approaches seek a geodesic curve passing through the mean that optimizes a criteria of interest. The requirements that the solution both is geodesic and must pass through the mean tend to imply that the methods only work well when the manifold is mostly flat within the support of the generating distribution. We argue that instead of generalizing linear Euclidean models, it is more fruitful to generalize non-linear Euclidean models. Specifically, we extend the classic Principal Curves from Hastie & Stuetzle to data residing on a complete Riemannian manifold. We show that for elliptical distributions in the tangent of spaces of constant curvature, the standard principal geodesic is a principal curve. The proposed model is simple to compute and avoids many of the pitfalls of traditional geodesic approaches. We empirically demonstrate the effectiveness of the Riemannian principal curves on several manifolds and datasets.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

PubMed

Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

2016-05-01

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria

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

PubMed

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.

Effectiveness of onsite wastewater reuse system in reducing bacterial contaminants measured with human-specific IMS/ATP and qPCR.

PubMed

Agidi, Senyo; Vedachalam, Sridhar; Mancl, Karen; Lee, Jiyoung

2013-01-30

Water shortages and the drive to recycle is increasing interest in reuse of reclaimed wastewater. Timely and cost-effective ways to detect fecal pollutants prior to reuse increases confidence of residents and neighbors concerned about reuse of reclaimed wastewater. The on-site wastewater treatment and reuse systems (OWTRS) used in this study include a septic tank, peat bioreactor, ClO(2) disinfection and land spray irrigation system. Bacteroides fragilis, Escherichia coli and Enterococcus spp., were tested with immunomagnetic separation/ATP bioluminescence (IMS/ATP), qPCR and culture-based methods. The results displayed a 2-log reduction in fecal bacteria in the peat bioreactor and a 5-log reduction following chloride dioxide disinfection. The fecal bacteria levels measured by IMS/ATP correlated with qPCR results: HuBac 16S (R(2) = 0.903), Bf-group 16S (R(2) = 0.956), gyrB (R(2) = 0.673), and Ent 23S (R(2) = 0.724). This is the first study in which the newly developed human-specific IMS/ATP and previously developed IMS/ATP were applied for determining OWTRS efficiency. Results of the study revealed that IMS/ATP is a timely and cost-effective way to detect fecal contaminants, and results were validated with qPCR and culture based methods. The new IMS/ATP can also be applied broadly in the detection of human-originated fecal contamination. Copyright © 2012 Elsevier Ltd. All rights reserved.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

PubMed Central

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. PMID:27195795

Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.

PubMed

Donia, D; Divizia, M; Pana', A

2005-06-01

Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.

Development and characterization of a synthetic DNA, NUversa, to be used as a standard in quantitative polymerase chain reactions for molecular pneumococcal serotyping.

PubMed

Sakai, Fuminori; Sonaty, Griffin; Watson, David; Klugman, Keith P; Vidal, Jorge E

2017-09-15

Identification of Streptococcus pneumoniae and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative polymerase chain reactions (qPCRs) have been developed for molecular detection, including a pan-pneumococcus lytA assay, and assays targeting 79 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study, we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes. Specificity of these new reactions was confirmed with a limit of detection between 2 and 20 genome equivalents/reaction. A synthetic DNA (NUversa, ∼8.2 kb) was then engineered to contain all available qPCR targets for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa (∼10.2 kb). Standards prepared from pNUversa and NUversa were compared against standards made out of genomic DNA. Linearity [NUversa (R2 > 0.982); pNUversa (R2 > 0.991)] and efficiency of qPCR reactions were similar to those utilizing chromosomal DNA (R2 > 0.981). Quantification with plasmid pNUversa was affected, however, whereas quantification with synthetic NUversa was comparable to that of genomic DNA. Therefore, NUversa may be utilized as DNA standard in single-plex assays of the currently known 94 pneumococcal serotypes. © FEMS 2017.

Determining the spill flow discharge of combined sewer overflows using rating curves based on computational fluid dynamics instead of the standard weir equation.

PubMed

Fach, S; Sitzenfrei, R; Rauch, W

2009-01-01

It is state of the art to evaluate and optimise sewer systems with urban drainage models. Since spill flow data is essential in the calibration process of conceptual models it is important to enhance the quality of such data. A wide spread approach is to calculate the spill flow volume by using standard weir equations together with measured water levels. However, these equations are only applicable to combined sewer overflow (CSO) structures, whose weir constructions correspond with the standard weir layout. The objective of this work is to outline an alternative approach to obtain spill flow discharge data based on measurements with a sonic depth finder. The idea is to determine the relation between water level and rate of spill flow by running a detailed 3D computational fluid dynamics (CFD) model. Two real world CSO structures have been chosen due to their complex structure, especially with respect to the weir construction. In a first step the simulation results were analysed to identify flow conditions for discrete steady states. It will be shown that the flow conditions in the CSO structure change after the spill flow pipe acts as a controlled outflow and therefore the spill flow discharge cannot be described with a standard weir equation. In a second step the CFD results will be used to derive rating curves which can be easily applied in everyday practice. Therefore the rating curves are developed on basis of the standard weir equation and the equation for orifice-type outlets. Because the intersection of both equations is not known, the coefficients of discharge are regressed from CFD simulation results. Furthermore, the regression of the CFD simulation results are compared with the one of the standard weir equation by using historic water levels and hydrographs generated with a hydrodynamic model. The uncertainties resulting of the wide spread use of the standard weir equation are demonstrated.

Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

PubMed

Åström, Johan; Pettersson, Thomas J R; Reischer, Georg H; Norberg, Tommy; Hermansson, Malte

2015-02-03

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

Incorporating Expert Judgments in Utility Evaluation of Bacteroidales qPCR Assays for Microbial Source Tracking in a Drinking Water Source

PubMed Central

Åström, Johan; Pettersson, Thomas J. R.; Reischer, Georg H.; Norberg, Tommy; Hermansson, Malte

2017-01-01

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays. PMID:25545113

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

EPA Science Inventory

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 ...

Lung function in North American Indian children: reference standards for spirometry, maximal expiratory flow volume curves, and peak expiratory flow.

PubMed

Wall, M A; Olson, D; Bonn, B A; Creelman, T; Buist, A S

1982-02-01

Reference standards of lung function was determined in 176 healthy North American Indian children (94 girls, 82 boys) 7 to 18 yr of age. Spirometry, maximal expiratory flow volume curves, and peak expiratory flow rate were measured using techniques and equipment recommended by the American Thoracic Society. Standing height was found to be an accurate predictor of lung function, and prediction equations for each lung function variable are presented using standing height as the independent variable. Lung volumes and expiratory flow rates in North American Indian children were similar to those previously reported for white and Mexican-American children but were greater than those in black children. In both boys and girls, lung function increased in a curvilinear fashion. Volume-adjusted maximal expiratory flow rates after expiring 50 or 75% of FVC tended to decrease in both sexes as age and height increased. Our maximal expiratory flow volume curve data suggest that as North American Indian children grow, lung volume increases at a slightly faster rate than airway size does.

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae.

PubMed

Martin-Sanchez, Pedro M; Gorbushina, Anna A; Kunte, Hans-Jörg; Toepel, Jörg

2016-07-01

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.

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

PubMed

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

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

PubMed

Horn, Ivo R; van Rijn, Menno; Zwetsloot, Tom J J; Basmagi, Said; Dirks-Mulder, Anita; van Leeuwen, Willem B; Ravensberg, Willem J; Gravendeel, Barbara

2016-02-01

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents. Copyright © 2015 Elsevier B.V. All rights reserved.

The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

PubMed

Cao, Yiping; Griffith, John F; Weisberg, Stephen B

2016-01-01

Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application.

On the analysis of Canadian Holstein dairy cow lactation curves using standard growth functions.

PubMed

López, S; France, J; Odongo, N E; McBride, R A; Kebreab, E; AlZahal, O; McBride, B W; Dijkstra, J

2015-04-01

Six classical growth functions (monomolecular, Schumacher, Gompertz, logistic, Richards, and Morgan) were fitted to individual and average (by parity) cumulative milk production curves of Canadian Holstein dairy cows. The data analyzed consisted of approximately 91,000 daily milk yield records corresponding to 122 first, 99 second, and 92 third parity individual lactation curves. The functions were fitted using nonlinear regression procedures, and their performance was assessed using goodness-of-fit statistics (coefficient of determination, residual mean squares, Akaike information criterion, and the correlation and concordance coefficients between observed and adjusted milk yields at several days in milk). Overall, all the growth functions evaluated showed an acceptable fit to the cumulative milk production curves, with the Richards equation ranking first (smallest Akaike information criterion) followed by the Morgan equation. Differences among the functions in their goodness-of-fit were enlarged when fitted to average curves by parity, where the sigmoidal functions with a variable point of inflection (Richards and Morgan) outperformed the other 4 equations. All the functions provided satisfactory predictions of milk yield (calculated from the first derivative of the functions) at different lactation stages, from early to late lactation. The Richards and Morgan equations provided the most accurate estimates of peak yield and total milk production per 305-d lactation, whereas the least accurate estimates were obtained with the logistic equation. In conclusion, classical growth functions (especially sigmoidal functions with a variable point of inflection) proved to be feasible alternatives to fit cumulative milk production curves of dairy cows, resulting in suitable statistical performance and accurate estimates of lactation traits. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

EPA Science Inventory

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...

Development and Evaluation of a Novel Curved Biopsy Device for CT-Guided Biopsy of Lesions Unreachable Using Standard Straight Needle Trajectories

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schulze-Hagen, Maximilian Franz, E-mail: mschulze@ukaachen.de; Pfeffer, Jochen; Zimmermann, Markus

PurposeTo evaluate the feasibility of a novel curved CT-guided biopsy needle prototype with shape memory to access otherwise not accessible biopsy targets.Methods and MaterialsA biopsy needle curved by 90° with specific radius was designed. It was manufactured using nitinol to acquire shape memory, encased in a straight guiding trocar to be driven out for access of otherwise inaccessible targets. Fifty CT-guided punctures were conducted in a biopsy phantom and 10 CT-guided punctures in a swine corpse. Biposies from porcine liver and muscle tissue were separately gained using the biopsy device, and histological examination was performed subsequently.ResultsMean time for placement ofmore » the trocar and deployment of the inner biopsy needle was ~205 ± 69 and ~93 ± 58 s, respectively, with a mean of ~4.5 ± 1.3 steps to reach adequate biopsy position. Mean distance from the tip of the needle to the target was ~0.7 ± 0.8 mm. CT-guided punctures in the swine corpse took relatively longer and required more biopsy steps (~574 ± 107 and ~380 ± 148 s, 8 ± 2.6 steps). Histology demonstrated appropriate tissue samples in nine out of ten cases (90%).ConclusionsTargets that were otherwise inaccessible via standard straight needle trajectories could be successfully reached with the curved biopsy needle prototype. Shape memory and preformed size with specific radius of the curved needle simplify the target accessibility with a low risk of injuring adjacent structures.« less

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Tracing Personalized Health Curves during Infections

PubMed Central

Schneider, David S.

2011-01-01

It is difficult to describe host–microbe interactions in a manner that deals well with both pathogens and mutualists. Perhaps a way can be found using an ecological definition of tolerance, where tolerance is defined as the dose response curve of health versus parasite load. To plot tolerance, individual infections are summarized by reporting the maximum parasite load and the minimum health for a population of infected individuals and the slope of the resulting curve defines the tolerance of the population. We can borrow this method of plotting health versus microbe load in a population and make it apply to individuals; instead of plotting just one point that summarizes an infection in an individual, we can plot the values at many time points over the course of an infection for one individual. This produces curves that trace the course of an infection through phase space rather than over a more typical timeline. These curves highlight relationships like recovery and point out bifurcations that are difficult to visualize with standard plotting techniques. Only nine archetypical curves are needed to describe most pathogenic and mutualistic host–microbe interactions. The technique holds promise as both a qualitative and quantitative approach to dissect host–microbe interactions of all kinds. PMID:21957398

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

PubMed

Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

2016-02-01

Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

lcps: Light curve pre-selection

NASA Astrophysics Data System (ADS)

Schlecker, Martin

2018-05-01

lcps searches for transit-like features (i.e., dips) in photometric data. Its main purpose is to restrict large sets of light curves to a number of files that show interesting behavior, such as drops in flux. While lcps is adaptable to any format of time series, its I/O module is designed specifically for photometry of the Kepler spacecraft. It extracts the pre-conditioned PDCSAP data from light curves files created by the standard Kepler pipeline. It can also handle csv-formatted ascii files. lcps uses a sliding window technique to compare a section of flux time series with its surroundings. A dip is detected if the flux within the window is lower than a threshold fraction of the surrounding fluxes.

Light curves for bump Cepheids computed with a dynamically zoned pulsation code

NASA Technical Reports Server (NTRS)

Adams, T. F.; Castor, J. I.; Davis, C. G.

1980-01-01

The dynamically zoned pulsation code developed by Castor, Davis, and Davison was used to recalculate the Goddard model and to calculate three other Cepheid models with the same period (9.8 days). This family of models shows how the bumps and other features of the light and velocity curves change as the mass is varied at constant period. The use of a code that is capable of producing reliable light curves demonstrates that the light and velocity curves for 9.8 day Cepheid models with standard homogeneous compositions do not show bumps like those that are observed unless the mass is significantly lower than the 'evolutionary mass.' The light and velocity curves for the Goddard model presented here are similar to those computed independently by Fischel, Sparks, and Karp. They should be useful as standards for future investigators.

Standard curves of placental weight and fetal/placental weight ratio in Japanese population: difference according to the delivery mode, fetal sex, or maternal parity.

PubMed

Ogawa, Masaki; Matsuda, Yoshio; Nakai, Akihito; Hayashi, Masako; Sato, Shoji; Matsubara, Shigeki

2016-11-01

Placental weight (PW) and fetal/placental weight ratio (F/P) have been considered to be useful parameters for understanding the pathophysiology of fetal growth. However, there have been no standard data on PW and F/P in Asian populations. This study was conducted to establish nomograms of PW and F/P in the Japanese population and to clarify characteristics of PW and F/P in this population. Included in the study were 79,590 Japanese cases: 58,871 vaginal and 20,719 cesarean deliveries at obstetrical facilities (2001-2002) and registered to the Japan Society of Obstetrics and Gynecology Database. Multiple pregnancies, stillbirths, and fetal anomalies were excluded. Nomograms of PW and F/P were created by spline methods in groups categorized by fetal sex (male or female) and maternal parity (primipara or multipara). Standard curves of PW and F/P were established, which indicated that PW and F/P were lower in cesarean deliveries than vaginal deliveries, especially during preterm period. PW differed depending on fetal sex and maternal parity. F/P differed according to fetal sex. We for the first time established standard curves of PW and F/P in the Japanese population with statistically sufficient data, which showed that PW and F/P were lower in cesarean deliveries. PW and F/P were also affected by fetal sex. These data might be useful to understand the pathophysiology between the fetus and placenta in utero. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Analysis of the curve of Spee and the curve of Wilson in adult Indian population: A three-dimensional measurement study.

PubMed

Surendran, Sowmya Velekkatt; Hussain, Sharmila; Bhoominthan, S; Nayar, Sanjna; Jayesh, Ragavendra

2016-01-01

When reconstructing the occlusal curvatures dentists often use a 4-inch radii arc as a rough standard based on Monson spherical theory. The use of an identical radius for the curve of Spee for all patients may not be appropriate because each patient is individually different. The validity of application of this theory in the Indian population and the present study has been undertaken. This study is an attempt to evaluate the curve of Spee and curve of Wilson in young Indian population using three dimensional analysis. This study compared the radius and the depth of right and left, maxillary and mandibular curves of Spee and the radius of maxillary and mandibular curves of Wilson in males and females. The cusp tips of canines, buccal cusp tips of premolars and molars and palatal/lingual cusp tips of second molars of 60 maxillary and 60 mandibular casts were obtained. Three-dimensional (x, y, z) coordinates of the cusp tips of the molars, premolars, and canines of the right and left sides of the maxilla and mandible were obtained with three dimensional coordinate measuring machine. The radius and the depth of right and left, maxillary and mandibular curves of Spee and the radius of maxillary and mandibular curves of Wilson were measured by means of computer software Metrologic-XG. Pearson's correlation test and Independent t-test were used to test the statistical significance (α=.05). The values of curve of Spee and curve of Wilson in Indian population obtained from this study were higher than the 4 inch (100 mm) radius proposed by Monson. These findings suggest ethnic differences in the radius of curve of Spee and curve of Wilson.

Anatomical curve identification

PubMed Central

Bowman, Adrian W.; Katina, Stanislav; Smith, Joanna; Brown, Denise

2015-01-01

Methods for capturing images in three dimensions are now widely available, with stereo-photogrammetry and laser scanning being two common approaches. In anatomical studies, a number of landmarks are usually identified manually from each of these images and these form the basis of subsequent statistical analysis. However, landmarks express only a very small proportion of the information available from the images. Anatomically defined curves have the advantage of providing a much richer expression of shape. This is explored in the context of identifying the boundary of breasts from an image of the female torso and the boundary of the lips from a facial image. The curves of interest are characterised by ridges or valleys. Key issues in estimation are the ability to navigate across the anatomical surface in three-dimensions, the ability to recognise the relevant boundary and the need to assess the evidence for the presence of the surface feature of interest. The first issue is addressed by the use of principal curves, as an extension of principal components, the second by suitable assessment of curvature and the third by change-point detection. P-spline smoothing is used as an integral part of the methods but adaptations are made to the specific anatomical features of interest. After estimation of the boundary curves, the intermediate surfaces of the anatomical feature of interest can be characterised by surface interpolation. This allows shape variation to be explored using standard methods such as principal components. These tools are applied to a collection of images of women where one breast has been reconstructed after mastectomy and where interest lies in shape differences between the reconstructed and unreconstructed breasts. They are also applied to a collection of lip images where possible differences in shape between males and females are of interest. PMID:26041943

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

EPA Science Inventory

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...

Methods for Performing Survival Curve Quality-of-Life Assessments.

PubMed

Sumner, Walton; Ding, Eric; Fischer, Irene D; Hagen, Michael D

2014-08-01

Many medical decisions involve an implied choice between alternative survival curves, typically with differing quality of life. Common preference assessment methods neglect this structure, creating some risk of distortions. Survival curve quality-of-life assessments (SQLA) were developed from Gompertz survival curves fitting the general population's survival. An algorithm was developed to generate relative discount rate-utility (DRU) functions from a standard survival curve and health state and an equally attractive alternative curve and state. A least means squared distance algorithm was developed to describe how nearly 3 or more DRU functions intersect. These techniques were implemented in a program called X-Trade and tested. SQLA scenarios can portray realistic treatment choices. A side effect scenario portrays one prototypical choice, to extend life while experiencing some loss, such as an amputation. A risky treatment scenario portrays procedures with an initial mortality risk. A time trade scenario mimics conventional time tradeoffs. Each SQLA scenario yields DRU functions with distinctive shapes, such as sigmoid curves or vertical lines. One SQLA can imply a discount rate or utility if the other value is known and both values are temporally stable. Two SQLA exercises imply a unique discount rate and utility if the inferred DRU functions intersect. Three or more SQLA results can quantify uncertainty or inconsistency in discount rate and utility estimates. Pilot studies suggested that many subjects could learn to interpret survival curves and do SQLA. SQLA confuse some people. Compared with SQLA, standard gambles quantify very low utilities more easily, and time tradeoffs are simpler for high utilities. When discount rates approach zero, time tradeoffs are as informative and easier to do than SQLA. SQLA may complement conventional utility assessment methods. © The Author(s) 2014.

Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos).

PubMed

Muradrasoli, Shaman; Mohamed, Nahla; Hornyák, Akos; Fohlman, Jan; Olsen, Björn; Belák, Sándor; Blomberg, Jonas

2009-08-01

Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

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

PubMed Central

Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

2015-01-01

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. PMID:26562020

Assessment of nitrification in groundwater filters for drinking water production by qPCR and activity measurement.

PubMed

de Vet, W W J M; Kleerebezem, R; van der Wielen, P W J J; Rietveld, L C; van Loosdrecht, M C M

2011-07-01

In groundwater treatment for drinking water production, the causes of nitrification problems and the effectiveness of process optimization in rapid sand filters are often not clear. To assess both issues, the performance of a full-scale groundwater filter with nitrification problems and another filter with complete nitrification and pretreatment by subsurface aeration was monitored over nine months. Quantitative real-time polymerase chain reaction (qPCR) targeting the amoA gene of bacteria and archaea and activity measurements of ammonia oxidation were used to regularly evaluate water and filter sand samples. Results demonstrated that subsurface aeration stimulated the growth of ammonia-oxidizing prokaryotes (AOP) in the aquifer. Cell balances, using qPCR counts of AOP for each filter, showed that the inoculated AOP numbers from the aquifer were marginal compared with AOP numbers detected in the filter. Excessive washout of AOP was not observed and did not cause the nitrification problems. Ammonia-oxidizing archaea grew in both filters, but only in low numbers compared to bacteria. The cell-specific nitrification rate in the sand and backwash water samples was high for the subsurface aerated filter, but systematically much lower for the filter with nitrification problems. From this, we conclude that incomplete nitrification was caused by nutrient limitation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting.

PubMed

Dehaut, Alexandre; Krzewinski, Frédéric; Grard, Thierry; Chollet, Marlène; Jacques, Philippe; Brisabois, Anne; Duflos, Guillaume

2016-04-01

Monitoring of early stages of freshness decay is a major issue for the fishery industry to guarantee the best quality for this highly perishable food matrix. Numerous techniques have been developed, but most of them have the disadvantage of being reliable only either in the last stages of fish freshness or for the analysis of whole fish. This study describes the development of a qPCR method targeting the torA gene harboured by fish spoilage microorganisms. torA encodes an enzyme that leads to the production of trimethylamine responsible for the characteristic spoiled-fish odour. A degenerate primer pair was designed. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies on 7-log DNA dilutions. The primer pair was used during a shelf-life monitoring study achieved on modified atmosphere packed, chilled, whiting (Merlangius merlangus) fillets. The qPCR approach allows the detection of an increase of torA copies throughout the storage of fillets in correlation with the evolution of both total volatile basic nitrogen (-0.86) and trimethylamine concentrations (-0.81), known as spoilage markers. This study described a very promising, sensitive, reliable, time-effective, technique in the field of freshness characterisation of processed fish. © 2015 Society of Chemical Industry.

Using Peano Curves to Construct Laplacians on Fractals

NASA Astrophysics Data System (ADS)

Molitor, Denali; Ott, Nadia; Strichartz, Robert

2015-12-01

We describe a new method to construct Laplacians on fractals using a Peano curve from the circle onto the fractal, extending an idea that has been used in the case of certain Julia sets. The Peano curve allows us to visualize eigenfunctions of the Laplacian by graphing the pullback to the circle. We study in detail three fractals: the pentagasket, the octagasket and the magic carpet. We also use the method for two nonfractal self-similar sets, the torus and the equilateral triangle, obtaining appealing new visualizations of eigenfunctions on the triangle. In contrast to the many familiar pictures of approximations to standard Peano curves, that do no show self-intersections, our descriptions of approximations to the Peano curves have self-intersections that play a vital role in constructing graph approximations to the fractal with explicit graph Laplacians that give the fractal Laplacian in the limit.

On curve veering and flutter of rotating blades

NASA Technical Reports Server (NTRS)

Afolabi, Dare; Mehmed, Oral

1993-01-01

The eigenvalues of rotating blades usually change with rotation speed according to the Stodola-Southwell criterion. Under certain circumstances, the loci of eigenvalues belonging to two distinct modes of vibration approach each other very closely, and it may appear as if the loci cross each other. However, our study indicates that the observable frequency loci of an undamped rotating blade do not cross, but must either repel each other (leading to 'curve veering'), or attract each other (leading to 'frequency coalescence'). Our results are reached by using standard arguments from algebraic geometry--the theory of algebraic curves and catastrophe theory. We conclude that it is important to resolve an apparent crossing of eigenvalue loci into either a frequency coalescence or a curve veering, because frequency coalescence is dangerous since it leads to flutter, whereas curve veering does not precipitate flutter and is, therefore, harmless with respect to elastic stability.

The Effects of Autocorrelation on the Curve-of-Factors Growth Model

ERIC Educational Resources Information Center

Murphy, Daniel L.; Beretvas, S. Natasha; Pituch, Keenan A.

2011-01-01

This simulation study examined the performance of the curve-of-factors model (COFM) when autocorrelation and growth processes were present in the first-level factor structure. In addition to the standard curve-of factors growth model, 2 new models were examined: one COFM that included a first-order autoregressive autocorrelation parameter, and a…

The Short- and Long-Run Marginal Cost Curve: A Pedagogical Note.

ERIC Educational Resources Information Center

Sexton, Robert L.; And Others

1993-01-01

Contends that the standard description of the relationship between the long-run marginal cost curve and the short-run marginal cost curve is often misleading and imprecise. Asserts that a sampling of college-level textbooks confirms this confusion. Provides a definition and instructional strategy that can be used to promote student understanding…

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

PubMed

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 × 10 1 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.

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

PubMed Central

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

Sketching Curves for Normal Distributions--Geometric Connections

ERIC Educational Resources Information Center

Bosse, Michael J.

2006-01-01

Within statistics instruction, students are often requested to sketch the curve representing a normal distribution with a given mean and standard deviation. Unfortunately, these sketches are often notoriously imprecise. Poor sketches are usually the result of missing mathematical knowledge. This paper considers relationships which exist among…

FECAL INDICATOR BACTERIA MEASUREMENTS BY QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS IN FRESH ARCHIVED DNA EXTRACT OF WATER SAMPLE FILTRATES

EPA Science Inventory

The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this stu...

quantGenius: implementation of a decision support system for qPCR-based gene quantification.

PubMed

Baebler, Špela; Svalina, Miha; Petek, Marko; Stare, Katja; Rotter, Ana; Pompe-Novak, Maruša; Gruden, Kristina

2017-05-25

Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification. We have developed "quantGenius" ( http://quantgenius.nib.si ), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry-independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases. To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

PubMed Central

Villarreal, Martha Lissete Morales; Padilha, Marina; Vieira, Antonio Diogo Silva; Franco, Bernadette Dora Gombossy de Melo; Martinez, Rafael Chacon Ruiz; Saad, Susana Marta Isay

2013-01-01

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress

Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil.

PubMed

Giglioti, R; Oliveira, H N; Santana, C H; Ibelli, A M G; Néo, T A; Bilhassi, T B; Rabelo, M D; Machado, R Z; Brito, L G; Oliveira, M C S

2016-07-01

The levels of infection by Babesia bovis and Babesia bigemina were estimated by absolute quantification through the quantitative PCR technique (qPCR). Fifty-one contemporaneous Angus cattle were evaluated on two occasions. The number of standard female Rhipicephalus microplus ticks present on the left side of the body was counted and blood samples were drawn from the tail vein into tubes containing the anticoagulant EDTA. The blood samples were submitted to DNA extraction and used to quantify the number of copies (NC) of DNA from B. bovis and B. bigemina by qPCR. The data on tick count and number of DNA copies were transformed for normalization and analyzed by a mixed model method. A multivariate model with repeated measures of the same animal, including the effects of collection, parasite species and their interaction, was used. The repeatability values were obtained from the matrix of (co)variances and were expressed for each species. The correlations between the counts of different species on the same animal, in the same collection or different collections, were also estimated. The results showed the qPCR could distinguish the two between infection by the two Babesia species. Infection levels by B. bovis and B. bigemina were detected in 100% and 98% of the animals, respectively. Significant differences were found (P<0.05) between the NC of the two Babesia species, B. bovis 1.49±0.07 vs. B. bigemina 0.82±0.06. Low repeatabilities were found for the counts of R. microplus and NC of B. bovis and B. bigemina: 0.05, 0.10 and 0.02, respectively. The correlations between R. microplus count and NC of B. bovis and B. bigemina were both very near zero. However, an association was observed between the NC of the two species, with a correlation coefficient of 0.30 for measures from the same collection. The absence of associations between the quantity of DNA from B. bovis and B. bigemina and the tick counts suggests that the variation of parasitemia by the hemoparasites did

Analytical Problems and Suggestions in the Analysis of Behavioral Economic Demand Curves.

PubMed

Yu, Jihnhee; Liu, Liu; Collins, R Lorraine; Vincent, Paula C; Epstein, Leonard H

2014-01-01

Behavioral economic demand curves (Hursh, Raslear, Shurtleff, Bauman, & Simmons, 1988) are innovative approaches to characterize the relationships between consumption of a substance and its price. In this article, we investigate common analytical issues in the use of behavioral economic demand curves, which can cause inconsistent interpretations of demand curves, and then we provide methodological suggestions to address those analytical issues. We first demonstrate that log transformation with different added values for handling zeros changes model parameter estimates dramatically. Second, demand curves are often analyzed using an overparameterized model that results in an inefficient use of the available data and a lack of assessment of the variability among individuals. To address these issues, we apply a nonlinear mixed effects model based on multivariate error structures that has not been used previously to analyze behavioral economic demand curves in the literature. We also propose analytical formulas for the relevant standard errors of derived values such as P max, O max, and elasticity. The proposed model stabilizes the derived values regardless of using different added increments and provides substantially smaller standard errors. We illustrate the data analysis procedure using data from a relative reinforcement efficacy study of simulated marijuana purchasing.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

PubMed

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

PubMed Central

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

ROC curves in clinical chemistry: uses, misuses, and possible solutions.

PubMed

Obuchowski, Nancy A; Lieber, Michael L; Wians, Frank H

2004-07-01

ROC curves have become the standard for describing and comparing the accuracy of diagnostic tests. Not surprisingly, ROC curves are used often by clinical chemists. Our aims were to observe how the accuracy of clinical laboratory diagnostic tests is assessed, compared, and reported in the literature; to identify common problems with the use of ROC curves; and to offer some possible solutions. We reviewed every original work using ROC curves and published in Clinical Chemistry in 2001 or 2002. For each article we recorded phase of the research, prospective or retrospective design, sample size, presence/absence of confidence intervals (CIs), nature of the statistical analysis, and major analysis problems. Of 58 articles, 31% were phase I (exploratory), 50% were phase II (challenge), and 19% were phase III (advanced) studies. The studies increased in sample size from phase I to III and showed a progression in the use of prospective designs. Most phase I studies were powered to assess diagnostic tests with ROC areas >/=0.70. Thirty-eight percent of studies failed to include CIs for diagnostic test accuracy or the CIs were constructed inappropriately. Thirty-three percent of studies provided insufficient analysis for comparing diagnostic tests. Other problems included dichotomization of the gold standard scale and inappropriate analysis of the equivalence of two diagnostic tests. We identify available software and make some suggestions for sample size determination, testing for equivalence in diagnostic accuracy, and alternatives to a dichotomous classification of a continuous-scale gold standard. More methodologic research is needed in areas specific to clinical chemistry.

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.

PubMed

Haugg, Anke M; Rennspiess, Dorit; zur Hausen, Axel; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. © 2014 UICC.

A Method for Molecular Dynamics on Curved Surfaces

PubMed Central

Paquay, Stefan; Kusters, Remy

2016-01-01

Dynamics simulations of constrained particles can greatly aid in understanding the temporal and spatial evolution of biological processes such as lateral transport along membranes and self-assembly of viruses. Most theoretical efforts in the field of diffusive transport have focused on solving the diffusion equation on curved surfaces, for which it is not tractable to incorporate particle interactions even though these play a crucial role in crowded systems. We show here that it is possible to take such interactions into account by combining standard constraint algorithms with the classical velocity Verlet scheme to perform molecular dynamics simulations of particles constrained to an arbitrarily curved surface. Furthermore, unlike Brownian dynamics schemes in local coordinates, our method is based on Cartesian coordinates, allowing for the reuse of many other standard tools without modifications, including parallelization through domain decomposition. We show that by applying the schemes to the Langevin equation for various surfaces, we obtain confined Brownian motion, which has direct applications to many biological and physical problems. Finally we present two practical examples that highlight the applicability of the method: 1) the influence of crowding and shape on the lateral diffusion of proteins in curved membranes; and 2) the self-assembly of a coarse-grained virus capsid protein model. PMID:27028633

A Method for Molecular Dynamics on Curved Surfaces

NASA Astrophysics Data System (ADS)

Paquay, Stefan; Kusters, Remy

2016-03-01

Dynamics simulations of constrained particles can greatly aid in understanding the temporal and spatial evolution of biological processes such as lateral transport along membranes and self-assembly of viruses. Most theoretical efforts in the field of diffusive transport have focussed on solving the diffusion equation on curved surfaces, for which it is not tractable to incorporate particle interactions even though these play a crucial role in crowded systems. We show here that it is possible to combine standard constraint algorithms with the classical velocity Verlet scheme to perform molecular dynamics simulations of particles constrained to an arbitrarily curved surface, in which such interactions can be taken into account. Furthermore, unlike Brownian dynamics schemes in local coordinates, our method is based on Cartesian coordinates allowing for the reuse of many other standard tools without modifications, including parallelisation through domain decomposition. We show that by applying the schemes to the Langevin equation for various surfaces, confined Brownian motion is obtained, which has direct applications to many biological and physical problems. Finally we present two practical examples that highlight the applicability of the method: (i) the influence of crowding and shape on the lateral diffusion of proteins in curved membranes and (ii) the self-assembly of a coarse-grained virus capsid protein model.

Effect of different breath alcohol concentrations on driving performance in horizontal curves.

PubMed

Zhang, Xingjian; Zhao, Xiaohua; Du, Hongji; Ma, Jianming; Rong, Jian

2014-11-01

Driving under the influence of alcohol on curved roadway segments has a higher risk than driving on straight segments. To explore the effect of different breath alcohol concentration (BrAC) levels on driving performance in roadway curves, a driving simulation experiment was designed to collect 25 participants' driving performance parameters (i.e., speed and lane position) under the influence of 4 BrAC levels (0.00%, 0.03%, 0.06% and 0.09%) on 6 types of roadway curves (3 radii×2 turning directions). Driving performance data for 22 participants were collected successfully. Then the average and standard deviation of the two parameters were analyzed, considering the entire curve and different sections of the curve, respectively. The results show that the speed throughout curves is higher when drinking and driving than during sober driving. The significant interaction between alcohol and radius exists in the middle and tangent segments after a curve exit, indicating that a small radius can reduce speed at high BrAC levels. The significant impairment of alcohol on the stability of speed occurs mainly in the curve section between the point of curve (PC) and point of tangent (PT), with no impairment noted in tangent sections. The stability of speed is significantly worsened at higher BrAC levels. Alcohol and radius have interactive effects on the standard deviation of speed in the entry segment of curves, indicating that the small radius amplifies the instability of speed at high BrAC levels. For lateral movement, drivers tend to travel on the right side of the lane when drinking and driving, mainly in the approach and middle segments of curves. Higher BrAC levels worsen the stability of lateral movement in every segment of the curve, regardless of its radius and turning direction. The results are expected to provide reference for detecting the drinking and driving state. Copyright © 2014 Elsevier Ltd. All rights reserved.

Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

PubMed

Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

2008-04-01

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Development of a rapid method to quantify Salmonella Typhimurium using a combination of MPN with qPCR and a shortened time incubation.

PubMed

Kim, Sun Ae; Park, Si Hong; Lee, Sang In; Ricke, Steven C

2017-08-01

A novel method was developed for the specific quantification of S. Typhimurium using a most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT). For S. Typhimurium enumeration, dilutions of samples were transferred into three wells on a microtiter plate and the plate was incubated for 4 h. The S. Typhimurium presence in the wells was identified using a qPCR and populations were determined based on an MPN calculation. The R 2 between the MPN-qPCR-SIT and conventional MPN exhibited a high level of correlation (0.9335-0.9752), suggesting that the MPN-qPCR-SIT offers a reliable alternative method for S. Typhimurium quantification. Although plating and qPCR were limited in their ability to detect low levels of S. Typhimurium (e.g. 0.18 log MPN/ml), these levels could be successfully detected with the MPN-qPCR-SIT. Chicken breast samples inoculated with S. Typhimurium were incubated at 0, 4, and 24 h and incubated samples were subjected to microbiome analysis. Levels of Salmonella and Enterobacteriaceae increased significantly with incubation time. The obvious benefits of the MPN-qPCR-SIT are: 1) a further confirmation step is not required, 2) the detection limit is as low as conventional MPN, but 3) is more rapid, requiring approximately 7 h to simultaneously complete quantification. Copyright © 2017 Elsevier Ltd. All rights reserved.

Density measurement of Demodex canis by qPCR and analysis of serum cytokine levels in dogs with different clinical forms of demodicosis.

PubMed

Gasparetto, Naiani Domingos; Almeida, Arleana do Bom Parto Ferreira; Nakazato, Luciano; França, Eduardo Luzía; França, Adenilda Cristina Honorio; Fagundes, Danny Laura Gomes; Bortolini, Juliano; Sousa, Valéria Régia Franco

2018-06-15

To quantify (by qPCR) the density of Demodex canis mites in the skin of dogs with demodicosis and in healthy dogs, as well as measuring the serum concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, and tumour necrosis factor-alfa (TNF-α). Fifty-four dogs were divided into three groups: localized demodicosis (LD, n = 16), generalized demodicosis (GD, n = 22), and control group (CG, n = 16). All dogs were subjected to skin scraping, blood collection, and skin biopsy. DNA extraction was performed and the parasite density was established by qPCR. Serum cytokine concentrations were obtained by flow cytometry. The median number of mites in the skin of the GD (6.2 × 10 4 copies/μL) and LD dogs (1.2 × 10 4 copies/μL) was statistically higher than that in the CG dogs (8.7 × 10 2 copies/μL). Whereas there were no significant differences in median IL-1β, IL-8, IL-10, IL-12, and TNF-α levels among the study groups, there was a statistically higher IL-6 concentration in the LD dogs than in the healthy dogs. According to our results, qPCR is an effective method for measuring the density of D. canis in the canine integument. In addition, the activation of the acute-phase immune response in localized demodicosis can be induced by IL-6 activity. Copyright © 2018 Elsevier B.V. All rights reserved.

Compression of contour data through exploiting curve-to-curve dependence

NASA Technical Reports Server (NTRS)

Yalabik, N.; Cooper, D. B.

1975-01-01

An approach to exploiting curve-to-curve dependencies in order to achieve high data compression is presented. One of the approaches to date of along curve compression through use of cubic spline approximation is taken and extended by investigating the additional compressibility achievable through curve-to-curve structure exploitation. One of the models under investigation is reported on.

Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices

PubMed Central

Sethulekshmi, C.; Latha, C.; Anu, C. J.

2018-01-01

Aim: The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices. Materials and Methods: A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry. Results: The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx 1 and stx 2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx 1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R2 of linear regression curves were −3.10, 34.24, and 0.99, respectively. Conclusion: The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food

Next-Generation Intensity-Duration-Frequency Curves for Hydrologic Design in Snow-Dominated Environments

NASA Astrophysics Data System (ADS)

Yan, Hongxiang; Sun, Ning; Wigmosta, Mark; Skaggs, Richard; Hou, Zhangshuan; Leung, Ruby

2018-02-01

There is a renewed focus on the design of infrastructure resilient to extreme hydrometeorological events. While precipitation-based intensity-duration-frequency (IDF) curves are commonly used as part of infrastructure design, a large percentage of peak runoff events in snow-dominated regions are caused by snowmelt, particularly during rain-on-snow (ROS) events. In these regions, precipitation-based IDF curves may lead to substantial overestimation/underestimation of design basis events and subsequent overdesign/underdesign of infrastructure. To overcome this deficiency, we proposed next-generation IDF (NG-IDF) curves, which characterize the actual water reaching the land surface. We compared NG-IDF curves to standard precipitation-based IDF curves for estimates of extreme events at 376 Snowpack Telemetry (SNOTEL) stations across the western United States that each had at least 30 years of high-quality records. We found standard precipitation-based IDF curves at 45% of the stations were subject to underdesign, many with significant underestimation of 100 year extreme events, for which the precipitation-based IDF curves can underestimate water potentially available for runoff by as much as 125% due to snowmelt and ROS events. The regions with the greatest potential for underdesign were in the Pacific Northwest, the Sierra Nevada Mountains, and the Middle and Southern Rockies. We also found the potential for overdesign at 20% of the stations, primarily in the Middle Rockies and Arizona mountains. These results demonstrate the need to consider snow processes in the development of IDF curves, and they suggest use of the more robust NG-IDF curves for hydrologic design in snow-dominated environments.

Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

PubMed

Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

2016-09-01

Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

Observational evidence of dust evolution in galactic extinction curves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cecchi-Pestellini, Cesare; Casu, Silvia; Mulas, Giacomo

Although structural and optical properties of hydrogenated amorphous carbons are known to respond to varying physical conditions, most conventional extinction models are basically curve fits with modest predictive power. We compare an evolutionary model of the physical properties of carbonaceous grain mantles with their determination by homogeneously fitting observationally derived Galactic extinction curves with the same physically well-defined dust model. We find that a large sample of observed Galactic extinction curves are compatible with the evolutionary scenario underlying such a model, requiring physical conditions fully consistent with standard density, temperature, radiation field intensity, and average age of diffuse interstellar clouds.more » Hence, through the study of interstellar extinction we may, in principle, understand the evolutionary history of the diffuse interstellar clouds.« less

Guiding curve based on the normal breathing as monitored by thermocouple for regular breathing.

PubMed

Lim, Sangwook; Park, Sung Ho; Ahn, Seung Do; Suh, Yelin; Shin, Seong Soo; Lee, Sang-wook; Kim, Jong Hoon; Choi, Eun Kyoung; Yi, Byong Yong; Kwon, Soo Il; Kim, Sookil; Jeung, Tae Sig

2007-11-01

Adapting radiation fields to a moving target requires information continuously on the location of internal target by detecting it directly or indirectly. The aim of this study is to make the breathing regular effectively with minimizing stress to the patient. A system for regulating patient's breath consists of a respiratory monitoring mask (ReMM), a thermocouple module, a screen, inner earphones, and a personal computer. A ReMM with thermocouple was developed previously to measure the patient's respiration. A software was written in LabView 7.0 (National Instruments, TX), which acquires respiration signal and displays its pattern. Two curves are displayed on the screen: One is a curve indicating the patient's current breathing pattern; the other is a guiding curve, which is iterated with one period of the patient's normal breathing curve. The guiding curves were acquired for each volunteer before they breathed with guidance. Ten volunteers participated in this study to evaluate this system. A cycle of the representative guiding curve was acquired by monitoring each volunteer's free breathing with ReMM and was then generated iteratively. The regularity was compared between a free breath curve and a guided breath curve by measuring standard deviations of amplitudes and periods of two groups of breathing. When the breathing was guided, the standard deviation of amplitudes and periods on average were reduced from 0.0029 to 0.00139 (arbitrary units) and from 0.359 s to 0.202 s, respectively. And the correlation coefficients between breathing curves and guiding curves were greater than 0.99 for all volunteers. The regularity was improved statistically when the guiding curve was used.

Learning micro incision surgery without the learning curve

PubMed Central

Navin, Shoba; Parikh, Rajul

2008-01-01

We describe a method of learning micro incision cataract surgery painlessly with the minimum of learning curves. A large-bore or standard anterior chamber maintainer (ACM) facilitates learning without change of machine or preferred surgical technique. Experience with the use of an ACM during phacoemulsification is desirable. PMID:18292624

Development of a tick-borne pathogen QPCR panel for detection of Anaplasma, Ehrlichia, Rickettsia, and Lyme disease Borrelia in animals.

PubMed

Shen, Zhenyu; Zhang, Michael Z; Stich, Roger W; Mitchell, William J; Zhang, Shuping

2018-05-23

Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals. Copyright © 2017. Published by Elsevier B.V.

Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum.

PubMed

Rubio, Marcela da Silva; Penha Filho, Rafael Antonio Casarin; Almeida, Adriana Maria de; Berchieri, Angelo

2017-12-01

Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10 8 -10 1 CFU of these biovars.

Next-Generation Intensity-Duration-Frequency Curves for Hydrologic Design in Snow-Dominated Environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hongxiang; Sun, Ning; Wigmosta, Mark

There is a renewed focus on the design of infrastructure resilient to extreme hydrometeorological events. While precipitation-based intensity-duration-frequency (IDF) curves are commonly used as part of infrastructure design, a large percentage of peak runoff events in snow-dominated regions are caused by snowmelt, particularly during rain-on-snow (ROS) events. In these regions, precipitation-based IDF curves may lead to substantial over-/under-estimation of design basis events and subsequent over-/under-design of infrastructure. To overcome this deficiency, we proposed next-generation IDF (NG-IDF) curves, which characterize the actual water reaching the land surface. We compared NG-IDF curves to standard precipitation-based IDF curves for estimates of extreme eventsmore » at 376 Snowpack Telemetry (SNOTEL) stations across the western United States that each had at least 30 years of high-quality records. We found standard precipitation-based IDF curves at 45% of the stations were subject to under-design, many with significant under-estimation of 100-year extreme events, for which the precipitation-based IDF curves can underestimate water potentially available for runoff by as much as 125% due to snowmelt and ROS events. The regions with the greatest potential for under-design were in the Pacific Northwest, the Sierra Nevada Mountains, and the Middle and Southern Rockies. We also found the potential for over-design at 20% of the stations, primarily in the Middle Rockies and Arizona mountains. These results demonstrate the need to consider snow processes in the development of IDF curves, and they suggest use of the more robust NG-IDF curves for hydrologic design in snow-dominated environments.« less

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Variation of curve number with storm depth

NASA Astrophysics Data System (ADS)

Banasik, K.; Hejduk, L.

2012-04-01

The NRCS Curve Number (known also as SCS-CN) method is well known as a tool in predicting flood runoff depth from small ungauged catchment. The traditional way of determination the CNs, based on soil characteristics, land use and hydrological conditions, seemed to have tendency to overpredict the floods in some cases. Over 30 year rainfall-runoff data, collected in two small (A=23.4 & 82.4 km2), lowland, agricultural catchments in Center of Poland (Banasik & Woodward 2010), were used to determine runoff Curve Number and to check a tendency of changing. The observed CN declines with increasing storm size, which according recent views of Hawkins (1993) could be classified as a standard response of watershed. The analysis concluded, that using CN value according to the procedure described in USDA-SCS Handbook one receives representative value for estimating storm runoff from high rainfall depths in the analyzes catchments. This has been confirmed by applying "asymptotic approach" for estimating the watershed curve number from the rainfall-runoff data. Furthermore, the analysis indicated that CN, estimated from mean retention parameter S of recorded events with rainfall depth higher than initial abstraction, is also approaching the theoretical CN. The observed CN, ranging from 59.8 to 97.1 and from 52.3 to 95.5, in the smaller and the larger catchment respectively, declines with increasing storm size, which has been classified as a standard response of watershed. The investigation demonstrated also changeability of the CN during a year, with much lower values during the vegetation season. Banasik K. & D.E. Woodward (2010). "Empirical determination of curve number for a small agricultural watrshed in Poland". 2nd Joint Federal Interagency Conference, Las Vegas, NV, June 27 - July 1, 2010 (http://acwi.gov/sos/pubs/2ndJFIC/Contents/10E_Banasik_ 28_02_10. pdf). Hawkins R. H. (1993). "Asymptotic determination of curve numbers from data". Journal of Irrigation and Drainage

Producing standard damaged DNA samples by heating: pitfalls and suggestions.

PubMed

Fattorini, Paolo; Marrubini, Giorgio; Bonin, Serena; Bertoglio, Barbara; Grignani, Pierangela; Recchia, Elisa; Pitacco, Paola; Procopio, Francesca; Cantoni, Carolina; Pajnič, Irena Zupanič; Sorçaburu-Cigliero, Solange; Previderè, Carlo

2018-05-15

Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0-18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ("Protocol P") and in Eppendorf tubes provided with screwcaps ("Protocol S"). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the "Protocol S" affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2-3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO 2 , ROS, NO x and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of

Master curve characterization of the fracture toughness behavior in SA508 Gr.4N low alloy steels

NASA Astrophysics Data System (ADS)

Lee, Ki-Hyoung; Kim, Min-Chul; Lee, Bong-Sang; Wee, Dang-Moon

2010-08-01

The fracture toughness properties of the tempered martensitic SA508 Gr.4N Ni-Mo-Cr low alloy steel for reactor pressure vessels were investigated by using the master curve concept. These results were compared to those of the bainitic SA508 Gr.3 Mn-Mo-Ni low alloy steel, which is a commercial RPV material. The fracture toughness tests were conducted by 3-point bending with pre-cracked charpy (PCVN) specimens according to the ASTM E1921-09c standard method. The temperature dependency of the fracture toughness was steeper than those predicted by the standard master curve, while the bainitic SA508 Gr.3 steel fitted well with the standard prediction. In order to properly evaluate the fracture toughness of the Gr.4N steels, the exponential coefficient of the master curve equation was changed and the modified curve was applied to the fracture toughness test results of model alloys that have various chemical compositions. It was found that the modified curve provided a better description for the overall fracture toughness behavior and adequate T0 determination for the tempered martensitic SA508 Gr.4N steels.

Quantifying and Reducing Curve-Fitting Uncertainty in Isc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campanelli, Mark; Duck, Benjamin; Emery, Keith

2015-06-14

Current-voltage (I-V) curve measurements of photovoltaic (PV) devices are used to determine performance parameters and to establish traceable calibration chains. Measurement standards specify localized curve fitting methods, e.g., straight-line interpolation/extrapolation of the I-V curve points near short-circuit current, Isc. By considering such fits as statistical linear regressions, uncertainties in the performance parameters are readily quantified. However, the legitimacy of such a computed uncertainty requires that the model be a valid (local) representation of the I-V curve and that the noise be sufficiently well characterized. Using more data points often has the advantage of lowering the uncertainty. However, more data pointsmore » can make the uncertainty in the fit arbitrarily small, and this fit uncertainty misses the dominant residual uncertainty due to so-called model discrepancy. Using objective Bayesian linear regression for straight-line fits for Isc, we investigate an evidence-based method to automatically choose data windows of I-V points with reduced model discrepancy. We also investigate noise effects. Uncertainties, aligned with the Guide to the Expression of Uncertainty in Measurement (GUM), are quantified throughout.« less

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method

EPA Science Inventory

A quantitative polymerase chain reaction (qPCR) method for the detection of entercocci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of wat...

Comparison of two methods to determine fan performance curves using computational fluid dynamics

NASA Astrophysics Data System (ADS)

Onma, Patinya; Chantrasmi, Tonkid

2018-01-01

This work investigates a systematic numerical approach that employs Computational Fluid Dynamics (CFD) to obtain performance curves of a backward-curved centrifugal fan. Generating the performance curves requires a number of three-dimensional simulations with varying system loads at a fixed rotational speed. Two methods were used and their results compared to experimental data. The first method incrementally changes the mass flow late through the inlet boundary condition while the second method utilizes a series of meshes representing the physical damper blade at various angles. The generated performance curves from both methods are compared with an experiment setup in accordance with the AMCA fan performance testing standard.

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

EPA Science Inventory

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...

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

PubMed

Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

PubMed Central

Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium “Candidatus Liberibacter asiaticus” (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer. PMID:29772016

Simplified method for creating a density-absorbed dose calibration curve for the low dose range from Gafchromic EBT3 film.

PubMed

Gotanda, Tatsuhiro; Katsuda, Toshizo; Gotanda, Rumi; Kuwano, Tadao; Akagawa, Takuya; Tanki, Nobuyoshi; Tabuchi, Akihiko; Shimono, Tetsunori; Kawaji, Yasuyuki

2016-01-01

Radiochromic film dosimeters have a disadvantage in comparison with an ionization chamber in that the dosimetry process is time-consuming for creating a density-absorbed dose calibration curve. The purpose of this study was the development of a simplified method of creating a density-absorbed dose calibration curve from radiochromic film within a short time. This simplified method was performed using Gafchromic EBT3 film with a low energy dependence and step-shaped Al filter. The simplified method was compared with the standard method. The density-absorbed dose calibration curves created using the simplified and standard methods exhibited approximately similar straight lines, and the gradients of the density-absorbed dose calibration curves were -32.336 and -33.746, respectively. The simplified method can obtain calibration curves within a much shorter time compared to the standard method. It is considered that the simplified method for EBT3 film offers a more time-efficient means of determining the density-absorbed dose calibration curve within a low absorbed dose range such as the diagnostic range.

Simplified method for creating a density-absorbed dose calibration curve for the low dose range from Gafchromic EBT3 film

PubMed Central

Gotanda, Tatsuhiro; Katsuda, Toshizo; Gotanda, Rumi; Kuwano, Tadao; Akagawa, Takuya; Tanki, Nobuyoshi; Tabuchi, Akihiko; Shimono, Tetsunori; Kawaji, Yasuyuki

2016-01-01

Radiochromic film dosimeters have a disadvantage in comparison with an ionization chamber in that the dosimetry process is time-consuming for creating a density-absorbed dose calibration curve. The purpose of this study was the development of a simplified method of creating a density-absorbed dose calibration curve from radiochromic film within a short time. This simplified method was performed using Gafchromic EBT3 film with a low energy dependence and step-shaped Al filter. The simplified method was compared with the standard method. The density-absorbed dose calibration curves created using the simplified and standard methods exhibited approximately similar straight lines, and the gradients of the density-absorbed dose calibration curves were −32.336 and −33.746, respectively. The simplified method can obtain calibration curves within a much shorter time compared to the standard method. It is considered that the simplified method for EBT3 film offers a more time-efficient means of determining the density-absorbed dose calibration curve within a low absorbed dose range such as the diagnostic range. PMID:28144120

Real-time image sequence segmentation using curve evolution

NASA Astrophysics Data System (ADS)

Zhang, Jun; Liu, Weisong

2001-04-01

In this paper, we describe a novel approach to image sequence segmentation and its real-time implementation. This approach uses the 3D structure tensor to produce a more robust frame difference signal and uses curve evolution to extract whole objects. Our algorithm is implemented on a standard PC running the Windows operating system with video capture from a USB camera that is a standard Windows video capture device. Using the Windows standard video I/O functionalities, our segmentation software is highly portable and easy to maintain and upgrade. In its current implementation on a Pentium 400, the system can perform segmentation at 5 frames/sec with a frame resolution of 160 by 120.

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

PubMed Central

Martins, António; Moreira, Cristiana; Vale, Micaela; Freitas, Marisa; Regueiras, Ana; Antunes, Agostinho; Vasconcelos, Vitor

2011-01-01

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. PMID:22072994

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

NASA Astrophysics Data System (ADS)

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.

Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

PubMed

Marques, Lívia Érika Carlos; Frota, Cristiane Cunha; Quetz, Josiane da Silva; Bindá, Alexandre Havt; Mota, Rosa Maria Salane; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Kendall, Carl; Kerr, Ligia Regina Franco Sansigolo

2017-12-26

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T m  = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10 3 -8.02 × 10 5 , and in SB samples from MB patients were 1.87 × 10 3 -1.50 × 10 6 . Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

PubMed

Uehara, Maiko; Tabata, Eri; Ishii, Kazuhiro; Sawa, Akira; Ohno, Misa; Sakaguchi, Masayoshi; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

2018-05-09

Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey ( Macaca fascicularis ) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

2012-08-01

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Infrared light curves of type Ia supernovae

DOE PAGES

Phillips, M. M.; Krisciunas, K.; Suntzeff, N. B.; ...

2003-10-02

This article provides a progress report on a collaborative program at the Las Campanas and Cerro Tololo Observatories to observe the near-IR light curves of Type Ia supernovae. We discuss how the morphologies of the JHK light curves change as a function of the decline rate parameter Δm 15 (B). Evidence is presented which indicates that the absolute magnitudes in the H band have little or no dependence on the decline rate, suggesting that SNe Ia may be nearly perfect cosmological standard candles in the near-IR. A preliminary Hubble diagram in the H band is presented and compared with amore » similar diagram in V for the same objects. Finally, observations of two peculiar supernovae, 1999ac and 2001ay, are briefly discussed.« less

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

PubMed Central

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. PMID:28591185

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments

PubMed Central

Kim, Hyerin; Kang, NaNa; An, KyuHyeon; Koo, JaeHyung; Kim, Min-Soo

2016-01-01

Design of high-quality primers for multiple target sequences is essential for qPCR experiments, but is challenging due to the need to consider both homology tests on off-target sequences and the same stringent filtering constraints on the primers. Existing web servers for primer design have major drawbacks, including requiring the use of BLAST-like tools for homology tests, lack of support for ranking of primers, TaqMan probes and simultaneous design of primers against multiple targets. Due to the large-scale computational overhead, the few web servers supporting homology tests use heuristic approaches or perform homology tests within a limited scope. Here, we describe the MRPrimerW, which performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, supports design of TaqMan probes and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly in a web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341 963 135 in silico validated primers covering 99% of human and mouse genes. Free access: http://MRPrimerW.com. PMID:27154272

Power of tests for comparing trend curves with application to national immunization survey (NIS).

PubMed

Zhao, Zhen

2011-02-28

To develop statistical tests for comparing trend curves of study outcomes between two socio-demographic strata across consecutive time points, and compare statistical power of the proposed tests under different trend curves data, three statistical tests were proposed. For large sample size with independent normal assumption among strata and across consecutive time points, the Z and Chi-square test statistics were developed, which are functions of outcome estimates and the standard errors at each of the study time points for the two strata. For small sample size with independent normal assumption, the F-test statistic was generated, which is a function of sample size of the two strata and estimated parameters across study period. If two trend curves are approximately parallel, the power of Z-test is consistently higher than that of both Chi-square and F-test. If two trend curves cross at low interaction, the power of Z-test is higher than or equal to the power of both Chi-square and F-test; however, at high interaction, the powers of Chi-square and F-test are higher than that of Z-test. The measurement of interaction of two trend curves was defined. These tests were applied to the comparison of trend curves of vaccination coverage estimates of standard vaccine series with National Immunization Survey (NIS) 2000-2007 data. Copyright © 2011 John Wiley & Sons, Ltd.

Effect of the Macintosh curved blade size on direct laryngoscopic view in edentulous patients.

PubMed

Kim, Hyerim; Chang, Jee-Eun; Han, Sung-Hee; Lee, Jung-Man; Yoon, Soohyuk; Hwang, Jin-Young

2018-01-01

In the present study, we compared the laryngoscopic view depending on the size of the Macintosh curved blade in edentulous patients. Thirty-five edentulous adult patients scheduled for elective surgery were included in the study. After induction of anesthesia, two direct laryngoscopies were performed alternately using a standard-sized Macintosh curved blade (No. 4 for men and No. 3 for women) and smaller-sized Macintosh curved blade (No. 3 for men and No. 2 for women). During direct laryngoscopy with each blade, two digital photographs of the lateral view were taken when the blade tip was placed in the valleculae; the laryngoscope was lifted to achieve the best laryngeal view. Then, the best laryngeal views were assessed using the percentage of glottic opening (POGO) score. On the photographs of the lateral view of direct laryngoscopy, the angles between the line extending along the laryngoscopic handle and the horizontal line were measured. The POGO score was improved with the smaller-sized blade compared with the standard-sized blade (87.3% [11.8%] vs. 71.3% [20.0%], P<0.001, respectively). The angles between the laryngoscopic handle and the horizontal line were greater with the smaller-sized blade compared to the standard-sized blade when the blade tip was placed on the valleculae and when the laryngoscope was lifted to achieve the best laryngeal view (both P<0.001). Compared to a standard-sized Macintosh blade, a smaller-sized Macintosh curved blade improved the laryngeal exposure in edentulous patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

PubMed

Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

2016-10-30

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine.

PubMed

Willenburg, Elize; Divol, Benoit

2012-11-15

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

PubMed

Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

2014-10-01

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture. © The American Society of Tropical Medicine and Hygiene.

Curved sensors for compact high-resolution wide-field designs: prototype demonstration and optical characterization

NASA Astrophysics Data System (ADS)

Chambion, Bertrand; Gaschet, Christophe; Behaghel, Thibault; Vandeneynde, Aurélie; Caplet, Stéphane; Gétin, Stéphane; Henry, David; Hugot, Emmanuel; Jahn, Wilfried; Lombardo, Simona; Ferrari, Marc

2018-02-01

Over the recent years, a huge interest has grown for curved electronics, particularly for opto-electronics systems. Curved sensors help the correction of off-axis aberrations, such as Petzval Field Curvature, astigmatism, and bring significant optical and size benefits for imaging systems. In this paper, we first describe advantages of curved sensor and associated packaging process applied on a 1/1.8'' format 1.3Mpx global shutter CMOS sensor (Teledyne EV76C560) into its standard ceramic package with a spherical radius of curvature Rc=65mm and 55mm. The mechanical limits of the die are discussed (Finite Element Modelling and experimental), and electro-optical performances are investigated. Then, based on the monocentric optical architecture, we proposed a new design, compact and with a high resolution, developed specifically for a curved image sensor including optical optimization, tolerances, assembly and optical tests. Finally, a functional prototype is presented through a benchmark approach and compared to an existing standard optical system with same performances and a x2.5 reduction of length. The finality of this work was a functional prototype demonstration on the CEA-LETI during Photonics West 2018 conference. All these experiments and optical results demonstrate the feasibility and high performances of systems with curved sensors.

A new form of the calibration curve in radiochromic dosimetry. Properties and results.

PubMed

Tamponi, Matteo; Bona, Rossana; Poggiu, Angela; Marini, Piergiorgio

2016-07-01

This work describes a new form of the calibration curve for radiochromic dosimetry that depends on one fit parameter. Some results are reported to show that the new curve performs as well as those previously used and, more importantly, significantly reduces the dependence on the lot of films, the film orientation on the scanner, and the time after exposure. The form of the response curve makes use of the net optical densities ratio against the dose and has been studied by means of the Beer-Lambert law and a simple modeling of the film. The new calibration curve has been applied to EBT3 films exposed at 6 and 15 MV energy beams of linear accelerators and read-out in transmission mode by means of a flatbed color scanner. Its performance has been compared to that of two established forms of the calibration curve, which use the optical density and the net optical density against the dose. Four series of measurements with four lots of EBT3 films were used to evaluate the precision, accuracy, and dependence on the time after exposure, orientation on the scanner and lot of films. The new calibration curve is roughly subject to the same dose uncertainty, about 2% (1 standard deviation), and has the same accuracy, about 1.5% (dose values between 50 and 450 cGy), as the other calibration curves when films of the same lot are used. Moreover, the new calibration curve, albeit obtained from only one lot of film, shows a good agreement with experimental data from all other lots of EBT3 films used, with an accuracy of about 2% and a relative dose precision of 2.4% (1 standard deviation). The agreement also holds for changes of the film orientation and of the time after exposure. The dose accuracy of this new form of the calibration curve is always equal to or better than those obtained from the two types of curves previously used. The use of the net optical densities ratio considerably reduces the dependence on the lot of films, the landscape/portrait orientation, and the time

Quantification of concentrated Chinese medicine granules by quantitative polymerase chain reaction.

PubMed

Lo, Yat-Tung; Shaw, Pang-Chui

2017-10-25

Determination of the amount of constituent in a multi-herb product is important for quality control. In concentrated Chinese medicine granules (CCMG), no dregs are left after dissolution of the CCMG. This study is the first to examine the feasibility of using quantitative polymerase chain reaction (qPCR) to find the amount of CCMG in solution form. DNA was extracted from Hirudo and Zaocys CCMG mixed at different ratios and amplified in qPCR using species-specific primers. The threshold cycle (C T ) obtained was compared with the respective standard curves. Results showed that reproducible quantification results could be obtained (1) for 5-50mg CCMG using a modified DNA extraction protocol, (2) amongst DNA extracted from the same batch of CCMG and (3) amongst different batches of CCMG from the same company. This study demonstrated the constitute amount of CCMG in a mixture could be determined using qPCR. This work has extended the application of DNA techniques for the quantification of herbal products and this approach may be developed for quality assurance in the CCMG industry. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment.

PubMed

Ates, Gamze; Mertens, Birgit; Heymans, Anja; Verschaeve, Luc; Milushev, Dimiter; Vanparys, Philippe; Roosens, Nancy H C; De Keersmaecker, Sigrid C J; Rogiers, Vera; Doktorova, Tatyana Y

2018-04-01

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG ™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG ™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

Data on regulation of the gene for the adipocyte-enriched micropeptide Adig/Smaf1 by qPCR analysis and luciferase reporter assay.

PubMed

Ren, Gang; Cairl, Nicholas; Kim, Ji Young; Smas, Cynthia M

2016-12-01

This article describes qPCR analysis for the Adig/Smaf1 gene in multiple in vitro adipocyte differentiation models including white and brown adipogenesis, cell lines and primary cultures. The article also contains qPCR data for transcript levels of Adig/Smaf1 in a wide panel of murine tissues. Expression of Adig/Smaf1 transcript in white and brown adipose tissue in fasted and refed mice is reported and also data for Adig/Smaf1 transcript expression in genetically obese ob/ob mice. Data on the effects of siRNA-mediated knockdown of Srebp1c on Adig/Smaf1 transcript levels in 3T3-L1 adipocytes are shown. Luciferase reporter assays provide data for regulation of an ~ 2 kb fragment of the 5' flanking region of Adig/Smaf1 gene by PPARγ/RXRα. This data is related to a research article describing Adig/Smaf1 protein expression, "Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes" (G. Ren, P. Eskandari, S. Wang, C.M. Smas, 2016) [1].

Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).

PubMed

Johansen, Ilona; Andreassen, Rune

2014-12-23

MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature

Quantifying and Reducing Curve-Fitting Uncertainty in Isc: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campanelli, Mark; Duck, Benjamin; Emery, Keith

Current-voltage (I-V) curve measurements of photovoltaic (PV) devices are used to determine performance parameters and to establish traceable calibration chains. Measurement standards specify localized curve fitting methods, e.g., straight-line interpolation/extrapolation of the I-V curve points near short-circuit current, Isc. By considering such fits as statistical linear regressions, uncertainties in the performance parameters are readily quantified. However, the legitimacy of such a computed uncertainty requires that the model be a valid (local) representation of the I-V curve and that the noise be sufficiently well characterized. Using more data points often has the advantage of lowering the uncertainty. However, more data pointsmore » can make the uncertainty in the fit arbitrarily small, and this fit uncertainty misses the dominant residual uncertainty due to so-called model discrepancy. Using objective Bayesian linear regression for straight-line fits for Isc, we investigate an evidence-based method to automatically choose data windows of I-V points with reduced model discrepancy. We also investigate noise effects. Uncertainties, aligned with the Guide to the Expression of Uncertainty in Measurement (GUM), are quantified throughout.« less

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays.

PubMed

Johansson, Anna H; Bejai, Sarosh; Niazi, Adnan; Manzoor, Shahid; Bongcam-Rudloff, Erik; Meijer, Johan

2014-12-01

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10(6)) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.

Stenting for curved lesions using a novel curved balloon: Preliminary experimental study.

PubMed

Tomita, Hideshi; Higaki, Takashi; Kobayashi, Toshiki; Fujii, Takanari; Fujimoto, Kazuto

2015-08-01

Stenting may be a compelling approach to dilating curved lesions in congenital heart diseases. However, balloon-expandable stents, which are commonly used for congenital heart diseases, are usually deployed in a straight orientation. In this study, we evaluated the effect of stenting with a novel curved balloon considered to provide better conformability to the curved-angled lesion. In vitro experiments: A Palmaz Genesis(®) stent (Johnson & Johnson, Cordis Co, Bridgewater, NJ, USA) mounted on the Goku(®) curve (Tokai Medical Co. Nagoya, Japan) was dilated in vitro to observe directly the behavior of the stent and balloon assembly during expansion. Animal experiment: A short Express(®) Vascular SD (Boston Scientific Co, Marlborough, MA, USA) stent and a long Express(®) Vascular LD stent (Boston Scientific) mounted on the curved balloon were deployed in the curved vessel of a pig to observe the effect of stenting in vivo. In vitro experiments: Although the stent was dilated in a curved fashion, stent and balloon assembly also rotated conjointly during expansion of its curved portion. In the primary stenting of the short stent, the stent was dilated with rotation of the curved portion. The excised stent conformed to the curved vessel. As the long stent could not be negotiated across the mid-portion with the balloon in expansion when it started curving, the mid-portion of the stent failed to expand fully. Furthermore, the balloon, which became entangled with the stent strut, could not be retrieved even after complete deflation. This novel curved balloon catheter might be used for implantation of the short stent in a curved lesion; however, it should not be used for primary stenting of the long stent. Post-dilation to conform the stent to the angled vessel would be safer than primary stenting irrespective of stent length. Copyright © 2014 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments.

PubMed

Kim, Hyerin; Kang, NaNa; An, KyuHyeon; Koo, JaeHyung; Kim, Min-Soo

2016-07-08

Design of high-quality primers for multiple target sequences is essential for qPCR experiments, but is challenging due to the need to consider both homology tests on off-target sequences and the same stringent filtering constraints on the primers. Existing web servers for primer design have major drawbacks, including requiring the use of BLAST-like tools for homology tests, lack of support for ranking of primers, TaqMan probes and simultaneous design of primers against multiple targets. Due to the large-scale computational overhead, the few web servers supporting homology tests use heuristic approaches or perform homology tests within a limited scope. Here, we describe the MRPrimerW, which performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, supports design of TaqMan probes and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly in a web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341 963 135 in silico validated primers covering 99% of human and mouse genes. Free access: http://MRPrimerW.com. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Are driving and overtaking on right curves more dangerous than on left curves?

PubMed

Othman, Sarbaz; Thomson, Robert; Lannér, Gunnar

2010-01-01

It is well known that crashes on horizontal curves are a cause for concern in all countries due to the frequency and severity of crashes at curves compared to road tangents. A recent study of crashes in western Sweden reported a higher rate of crashes in right curves than left curves. To further understand this result, this paper reports the results of novel analyses of the responses of vehicles and drivers during negotiating and overtaking maneuvers on curves for right hand traffic. The overall objectives of the study were to find road parameters for curves that affect vehicle dynamic responses, to analyze these responses during overtaking maneuvers on curves, and to link the results with driver behavior for different curve directions. The studied road features were speed, super-elevation, radius and friction including their interactions, while the analyzed vehicle dynamic factors were lateral acceleration and yaw angular velocity. A simulation program, PC-Crash, has been used to simulate road parameters and vehicle response interaction in curves. Overtaking maneuvers have been simulated for all road feature combinations in a total of 108 runs. Analysis of variances (ANOVA) was performed, using two sided randomized block design, to find differences in vehicle responses for the curve parameters. To study driver response, a field test using an instrumented vehicle and 32 participants was reviewed as it contained longitudinal speed and acceleration data for analysis. The simulation results showed that road features affect overtaking performance in right and left curves differently. Overtaking on right curves was sensitive to radius and the interaction of radius with road condition; while overtaking on left curves was more sensitive to super-elevation. Comparisons of lateral acceleration and yaw angular velocity during these maneuvers showed different vehicle response configurations depending on curve direction and maneuver path. The field test experiments also showed

It's time to move on from the bell curve.

PubMed

Robinson, Lawrence R

2017-11-01

The bell curve was first described in the 18th century by de Moivre and Gauss to depict the distribution of binomial events, such as coin tossing, or repeated measures of physical objects. In the 19th and 20th centuries, the bell curve was appropriated, or perhaps misappropriated, to apply to biologic and social measures across people. For many years we used it to derive reference values for our electrophysiologic studies. There is, however, no reason to believe that electrophysiologic measures should approximate a bell-curve distribution, and empiric evidence suggests they do not. The concept of using mean ± 2 standard deviations should be abandoned. Reference values are best derived by using non-parametric analyses, such as percentile values. This proposal aligns with the recommendation of the recent normative data task force of the American Association of Neuromuscular & Electrodiagnostic Medicine and follows sound statistical principles. Muscle Nerve 56: 859-860, 2017. © 2017 Wiley Periodicals, Inc.

Curve Boxplot: Generalization of Boxplot for Ensembles of Curves.

PubMed

Mirzargar, Mahsa; Whitaker, Ross T; Kirby, Robert M

2014-12-01

In simulation science, computational scientists often study the behavior of their simulations by repeated solutions with variations in parameters and/or boundary values or initial conditions. Through such simulation ensembles, one can try to understand or quantify the variability or uncertainty in a solution as a function of the various inputs or model assumptions. In response to a growing interest in simulation ensembles, the visualization community has developed a suite of methods for allowing users to observe and understand the properties of these ensembles in an efficient and effective manner. An important aspect of visualizing simulations is the analysis of derived features, often represented as points, surfaces, or curves. In this paper, we present a novel, nonparametric method for summarizing ensembles of 2D and 3D curves. We propose an extension of a method from descriptive statistics, data depth, to curves. We also demonstrate a set of rendering and visualization strategies for showing rank statistics of an ensemble of curves, which is a generalization of traditional whisker plots or boxplots to multidimensional curves. Results are presented for applications in neuroimaging, hurricane forecasting and fluid dynamics.

Percentile curves for skinfold thickness for Canadian children and youth.

PubMed

Kuhle, Stefan; Ashley-Martin, Jillian; Maguire, Bryan; Hamilton, David C

2016-01-01

Background. Skinfold thickness (SFT) measurements are a reliable and feasible method for assessing body fat in children but their use and interpretation is hindered by the scarcity of reference values in representative populations of children. The objective of the present study was to develop age- and sex-specific percentile curves for five SFT measures (biceps, triceps, subscapular, suprailiac, medial calf) in a representative population of Canadian children and youth. Methods. We analyzed data from 3,938 children and adolescents between 6 and 19 years of age who participated in the Canadian Health Measures Survey cycles 1 (2007/2009) and 2 (2009/2011). Standardized procedures were used to measure SFT. Age- and sex-specific centiles for SFT were calculated using the GAMLSS method. Results. Percentile curves were materially different in absolute value and shape for boys and girls. Percentile girls in girls steadily increased with age whereas percentile curves in boys were characterized by a pubertal centered peak. Conclusions. The current study has presented for the first time percentile curves for five SFT measures in a representative sample of Canadian children and youth.

Creation of three-dimensional craniofacial standards from CBCT images

NASA Astrophysics Data System (ADS)

Subramanyan, Krishna; Palomo, Martin; Hans, Mark

2006-03-01

Low-dose three-dimensional Cone Beam Computed Tomography (CBCT) is becoming increasingly popular in the clinical practice of dental medicine. Two-dimensional Bolton Standards of dentofacial development are routinely used to identify deviations from normal craniofacial anatomy. With the advent of CBCT three dimensional imaging, we propose a set of methods to extend these 2D Bolton Standards to anatomically correct surface based 3D standards to allow analysis of morphometric changes seen in craniofacial complex. To create 3D surface standards, we have implemented series of steps. 1) Converting bi-plane 2D tracings into set of splines 2) Converting the 2D splines curves from bi-plane projection into 3D space curves 3) Creating labeled template of facial and skeletal shapes and 4) Creating 3D average surface Bolton standards. We have used datasets from patients scanned with Hitachi MercuRay CBCT scanner providing high resolution and isotropic CT volume images, digitized Bolton Standards from age 3 to 18 years of lateral and frontal male, female and average tracings and converted them into facial and skeletal 3D space curves. This new 3D standard will help in assessing shape variations due to aging in young population and provide reference to correct facial anomalies in dental medicine.

Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients.

PubMed

Alanio, A; Desoubeaux, G; Sarfati, C; Hamane, S; Bergeron, A; Azoulay, E; Molina, J M; Derouin, F; Menotti, J

2011-10-01

Diagnosis of pneumocystosis usually relies on microscopic demonstration of Pneumocystis jirovecii in respiratory samples. Conventional PCR can detect low levels of P. jirovecii DNA but cannot differentiate active pneumonia from colonization. In this study, we used a new real-time quantitative PCR (qPCR) assay to identify and discriminate these entities. One hundred and sixty-three bronchoalveolar lavage fluids and 115 induced sputa were prospectively obtained from 238 consecutive immunocompromised patients presenting signs of pneumonia. Each patient was classified as having a high or a low probability of P. jirovecii pneumonia according to clinical and radiological presentation. Samples were processed by microscopy and by a qPCR assay amplifying the P. jirovecii mitochondrial large-subunit rRNA gene; qPCR results were expressed as trophic form equivalents (TFEq)/mL by reference to a standard curve obtained from numbered suspensions of trophic forms. From 21 samples obtained from 16 patients with a high probability of P. jirovecii pneumonia, 21 were positive by qPCR whereas only 16 were positive by microscopy. Fungal load ranged from 134 to 1.73 × 10(6)  TFEq/mL. Among 257 specimens sampled from 222 patients with a low probability of P. jirovecii pneumonia, 222 were negative by both techniques but 35 were positive by qPCR (0.1-1840 TFEq/mL), suggesting P. jirovecii colonization. Two cut-off values of 120 and 1900 TFEq/mL were proposed to discriminate active pneumonia from colonization, with a grey zone between them. In conclusion, this qPCR assay discriminates active pneumonia from colonization. This is particularly relevant for patient management, especially in non-human immunodeficiency virus (HIV)-infected immunocompromised patients, who often present low-burden P. jirovecii infections that are not diagnosed microscopically. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Rotation curve for the Milky Way galaxy in conformal gravity

NASA Astrophysics Data System (ADS)

O'Brien, James G.; Moss, Robert J.

2015-05-01

Galactic rotation curves have proven to be the testing ground for dark matter bounds in galaxies, and our own Milky Way is one of many large spiral galaxies that must follow the same models. Over the last decade, the rotation of the Milky Way galaxy has been studied and extended by many authors. Since the work of conformal gravity has now successfully fit the rotation curves of almost 140 galaxies, we present here the fit to our own Milky Way. However, the Milky Way is not just an ordinary galaxy to append to our list, but instead provides a robust test of a fundamental difference of conformal gravity rotation curves versus standard cold dark matter models. It was shown by Mannheim and O'Brien that in conformal gravity, the presence of a quadratic potential causes the rotation curve to eventually fall off after its flat portion. This effect can currently be seen in only a select few galaxies whose rotation curve is studied well beyond a few multiples of the optical galactic scale length. Due to the recent work of Sofue et al and Kundu et al, the rotation curve of the Milky Way has now been studied to a degree where we can test the predicted fall off in the conformal gravity rotation curve. We find that - like the other galaxies already studied in conformal gravity - we obtain amazing agreement with rotational data and the prediction includes the eventual fall off at large distances from the galactic center.

Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model

PubMed Central

López, Luisa F.; Muñoz, César O.; Cáceres, Diego H.; Tobón, Ángela M.; Loparev, Vladimir; Clay, Oliver; Chiller, Tom; Litvintseva, Anastasia; Gade, Lalitha; González, Ángel

2017-01-01

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis. PMID

Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model.

PubMed

López, Luisa F; Muñoz, César O; Cáceres, Diego H; Tobón, Ángela M; Loparev, Vladimir; Clay, Oliver; Chiller, Tom; Litvintseva, Anastasia; Gade, Lalitha; González, Ángel; Gómez, Beatriz L

2017-01-01

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.

Multi-laboratory survey of qPCR enterococci analysis method performance

EPA Pesticide Factsheets

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries fr

A new form of the calibration curve in radiochromic dosimetry. Properties and results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tamponi, Matteo, E-mail: mtamponi@aslsassari.it; B

Purpose: This work describes a new form of the calibration curve for radiochromic dosimetry that depends on one fit parameter. Some results are reported to show that the new curve performs as well as those previously used and, more importantly, significantly reduces the dependence on the lot of films, the film orientation on the scanner, and the time after exposure. Methods: The form of the response curve makes use of the net optical densities ratio against the dose and has been studied by means of the Beer–Lambert law and a simple modeling of the film. The new calibration curve hasmore » been applied to EBT3 films exposed at 6 and 15 MV energy beams of linear accelerators and read-out in transmission mode by means of a flatbed color scanner. Its performance has been compared to that of two established forms of the calibration curve, which use the optical density and the net optical density against the dose. Four series of measurements with four lots of EBT3 films were used to evaluate the precision, accuracy, and dependence on the time after exposure, orientation on the scanner and lot of films. Results: The new calibration curve is roughly subject to the same dose uncertainty, about 2% (1 standard deviation), and has the same accuracy, about 1.5% (dose values between 50 and 450 cGy), as the other calibration curves when films of the same lot are used. Moreover, the new calibration curve, albeit obtained from only one lot of film, shows a good agreement with experimental data from all other lots of EBT3 films used, with an accuracy of about 2% and a relative dose precision of 2.4% (1 standard deviation). The agreement also holds for changes of the film orientation and of the time after exposure. Conclusions: The dose accuracy of this new form of the calibration curve is always equal to or better than those obtained from the two types of curves previously used. The use of the net optical densities ratio considerably reduces the dependence on the lot of films

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

PubMed

Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

PubMed Central

Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

Development and validation of a novel hydrolysis probe real-time polymerase chain reaction for agamid adenovirus 1 in the central bearded dragon (Pogona vitticeps).

PubMed

Fredholm, Daniel V; Coleman, James K; Childress, April L; Wellehan, James F X

2015-03-01

Agamid adenovirus 1 (AgAdv-1) is a significant cause of disease in bearded dragons (Pogona sp.). Clinical manifestations of AgAdv-1 infection are variable and often nonspecific; the manifestations range from lethargy, weight loss, and inappetence, to severe enteritis, hepatitis, and sudden death. Currently, diagnosis of AgAdv-1 infection is achieved through a single published method: standard nested polymerase chain reaction (nPCR) and sequencing. Standard nPCR with sequencing provides reliable sensitivity, specificity, and validation of PCR products. However, this process is comparatively expensive, laborious, and slow. Probe hybridization, as used in a TaqMan assay, represents the best option for validating PCR products aside from the time-consuming process of sequencing. This study developed a real-time PCR (qPCR) assay using a TaqMan probe-based assay, targeting a highly conserved region of the AgAdv-1 genome. Standard curves were generated, detection results were compared with the gold standard conventional PCR and sequencing assay, and limits of detection were determined. Additionally, the qPCR assay was run on samples known to be positive for AgAdv-1 and samples known to be positive for other adenoviruses. Based on the results of these evaluations, this assay allows for a less expensive, rapid, quantitative detection of AgAdv-1 in bearded dragons. © 2015 The Author(s).

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

NASA Technical Reports Server (NTRS)

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.

Computerised curve deconvolution of TL/OSL curves using a popular spreadsheet program.

PubMed

Afouxenidis, D; Polymeris, G S; Tsirliganis, N C; Kitis, G

2012-05-01

This paper exploits the possibility of using commercial software for thermoluminescence and optically stimulated luminescence curve deconvolution analysis. The widely used software package Microsoft Excel, with the Solver utility has been used to perform deconvolution analysis to both experimental and reference glow curves resulted from the GLOw Curve ANalysis INtercomparison project. The simple interface of this programme combined with the powerful Solver utility, allows the analysis of complex stimulated luminescence curves into their components and the evaluation of the associated luminescence parameters.

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

PubMed

Kline, Margaret C; Duewer, David L; Travis, John C; Smith, Melody V; Redman, Janette W; Vallone, Peter M; Decker, Amy E; Butler, John M

2009-06-01

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

Percentile curves for skinfold thickness for Canadian children and youth

PubMed Central

Ashley-Martin, Jillian; Maguire, Bryan; Hamilton, David C.

2016-01-01

Background. Skinfold thickness (SFT) measurements are a reliable and feasible method for assessing body fat in children but their use and interpretation is hindered by the scarcity of reference values in representative populations of children. The objective of the present study was to develop age- and sex-specific percentile curves for five SFT measures (biceps, triceps, subscapular, suprailiac, medial calf) in a representative population of Canadian children and youth. Methods. We analyzed data from 3,938 children and adolescents between 6 and 19 years of age who participated in the Canadian Health Measures Survey cycles 1 (2007/2009) and 2 (2009/2011). Standardized procedures were used to measure SFT. Age- and sex-specific centiles for SFT were calculated using the GAMLSS method. Results. Percentile curves were materially different in absolute value and shape for boys and girls. Percentile girls in girls steadily increased with age whereas percentile curves in boys were characterized by a pubertal centered peak. Conclusions. The current study has presented for the first time percentile curves for five SFT measures in a representative sample of Canadian children and youth. PMID:27547554

Using Machine Learning To Predict Which Light Curves Will Yield Stellar Rotation Periods

NASA Astrophysics Data System (ADS)

Agüeros, Marcel; Teachey, Alexander

2018-01-01

Using time-domain photometry to reliably measure a solar-type star's rotation period requires that its light curve have a number of favorable characteristics. The probability of recovering a period will be a non-linear function of these light curve features, which are either astrophysical in nature or set by the observations. We employ standard machine learning algorithms (artificial neural networks and random forests) to predict whether a given light curve will produce a robust rotation period measurement from its Lomb-Scargle periodogram. The algorithms are trained and validated using salient statistics extracted from both simulated light curves and their corresponding periodograms, and we apply these classifiers to the most recent Intermediate Palomar Transient Factory (iPTF) data release. With this pipeline, we anticipate measuring rotation periods for a significant fraction of the ∼4x108 stars in the iPTF footprint.

Time Alignment as a Necessary Step in the Analysis of Sleep Probabilistic Curves

NASA Astrophysics Data System (ADS)

Rošt'áková, Zuzana; Rosipal, Roman

2018-02-01

Sleep can be characterised as a dynamic process that has a finite set of sleep stages during the night. The standard Rechtschaffen and Kales sleep model produces discrete representation of sleep and does not take into account its dynamic structure. In contrast, the continuous sleep representation provided by the probabilistic sleep model accounts for the dynamics of the sleep process. However, analysis of the sleep probabilistic curves is problematic when time misalignment is present. In this study, we highlight the necessity of curve synchronisation before further analysis. Original and in time aligned sleep probabilistic curves were transformed into a finite dimensional vector space, and their ability to predict subjects' age or daily measures is evaluated. We conclude that curve alignment significantly improves the prediction of the daily measures, especially in the case of the S2-related sleep states or slow wave sleep.

Optimization of curved drift tubes for ultraviolet-ion mobility spectrometry

NASA Astrophysics Data System (ADS)

Ni, Kai; Ou, Guangli; Zhang, Xiaoguo; Yu, Zhou; Yu, Quan; Qian, Xiang; Wang, Xiaohao

2015-08-01

Ion mobility spectrometry (IMS) is a key trace detection technique for toxic pollutants and explosives in the atmosphere. Ultraviolet radiation photoionization source is widely used as an ionization source for IMS due to its advantages of high selectivity and non-radioactivity. However, UV-IMS bring problems that UV rays will be launched into the drift tube which will cause secondary ionization and lead to the photoelectric effect of the Faraday disk. So air is often used as working gas to reduce the effective distance of UV rays, but it will limit the application areas of UV-IMS. In this paper, we propose a new structure of curved drift tube, which can avoid abnormally incident UV rays. Furthermore, using curved drift tube may increase the length of drift tube and then improve the resolution of UV-IMS according to previous research. We studied the homogeneity of electric field in the curved drift tube, which determined the performance of UV-IMS. Numerical simulation of electric field in curved drift tube was conducted by SIMION in our study. In addition, modeling method and homogeneity standard for electric field were also presented. The influences of key parameters include radius of gyration, gap between electrode as well as inner diameter of curved drift tube, on the homogeneity of electric field were researched and some useful laws were summarized. Finally, an optimized curved drift tube is designed to achieve homogenous drift electric field. There is more than 98.75% of the region inside the curved drift tube where the fluctuation of the electric field strength along the radial direction is less than 0.2% of that along the axial direction.

Differentiation of five enterohepatic Helicobacter species by nested PCR with high-resolution melting curve analysis.

PubMed

Wu, Miaoli; Rao, Dan; Zhu, Yujun; Wang, Jing; Yuan, Wen; Zhang, Yu; Huang, Ren; Guo, Pengju

2017-04-01

Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species. © 2016 John Wiley & Sons Ltd.

A quantitative PCR method for assessing the presence of Pasteurella testudinis DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

PubMed

duPre', S A; Tracy, C R; Sandmeier, F C; Hunter, K W

2012-12-01

Pasteurella testudinis has been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Our goal was to develop a sensitive and specific qPCR method for detecting DNA from P. testudinis in nasal lavage fluid collected from desert tortoises in the field. Probes for 16S ribosomal RNA and RNA polymerase β-subunit (rpoB) genes were designed. A standard curve generated with DNA extracted from known numbers of bacterial cells determined by flow cytometry revealed a lower detection limit of 50 fg/ml (10 bacteria/ml). The nasal lavage fluid contained no interfering substances, and the qPCR method did not recognize normal flora DNA. The nasal lavage samples from 20 desert tortoises captured in Clark County, Nevada, USA in 2007 and housed at the Desert Tortoise Conservation Center, were all positive for P. testudinis DNA by qPCR. Another set of 19 lavage samples collected in 2010 from wild desert tortoises in the Mojave Desert were tested and 84% were positive for P. testudinis DNA. Fully validated, this qPCR method will provide a means of determining colonization rate. When used in conjunction with serological methods and clinical evaluations, both infection rate and disease rate can be determined for this potential URTD pathogen. This new assay provides an important tool for managing the threatened populations of the Mojave Desert tortoise. Copyright © 2012 Elsevier B.V. All rights reserved.

Modeling error distributions of growth curve models through Bayesian methods.

PubMed

Zhang, Zhiyong

2016-06-01

Growth curve models are widely used in social and behavioral sciences. However, typical growth curve models often assume that the errors are normally distributed although non-normal data may be even more common than normal data. In order to avoid possible statistical inference problems in blindly assuming normality, a general Bayesian framework is proposed to flexibly model normal and non-normal data through the explicit specification of the error distributions. A simulation study shows when the distribution of the error is correctly specified, one can avoid the loss in the efficiency of standard error estimates. A real example on the analysis of mathematical ability growth data from the Early Childhood Longitudinal Study, Kindergarten Class of 1998-99 is used to show the application of the proposed methods. Instructions and code on how to conduct growth curve analysis with both normal and non-normal error distributions using the the MCMC procedure of SAS are provided.

The S-curve for forecasting waste generation in construction projects.

PubMed

Lu, Weisheng; Peng, Yi; Chen, Xi; Skitmore, Martin; Zhang, Xiaoling

2016-10-01

Forecasting construction waste generation is the yardstick of any effort by policy-makers, researchers, practitioners and the like to manage construction and demolition (C&D) waste. This paper develops and tests an S-curve model to indicate accumulative waste generation as a project progresses. Using 37,148 disposal records generated from 138 building projects in Hong Kong in four consecutive years from January 2011 to June 2015, a wide range of potential S-curve models are examined, and as a result, the formula that best fits the historical data set is found. The S-curve model is then further linked to project characteristics using artificial neural networks (ANNs) so that it can be used to forecast waste generation in future construction projects. It was found that, among the S-curve models, cumulative logistic distribution is the best formula to fit the historical data. Meanwhile, contract sum, location, public-private nature, and duration can be used to forecast construction waste generation. The study provides contractors with not only an S-curve model to forecast overall waste generation before a project commences, but also with a detailed baseline to benchmark and manage waste during the course of construction. The major contribution of this paper is to the body of knowledge in the field of construction waste generation forecasting. By examining it with an S-curve model, the study elevates construction waste management to a level equivalent to project cost management where the model has already been readily accepted as a standard tool. Copyright © 2016 Elsevier Ltd. All rights reserved.

Next-Generation Intensity-Duration-Frequency Curves for Hydrologic Design in Snow-Dominated Environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hongxiang; Sun, Ning; Wigmosta, Mark

Precipitation-based intensity-duration-frequency (PREC-IDF) curves are a standard tool used to derive design floods for hydraulic infrastructure worldwide. In snow-dominated regions where a large percentage of flood events are caused by snowmelt and rain-on-snow events, the PREC-IDF design approach can lead to substantial underestimation/overestimation of design floods and associated infrastructure. In this study, next-generation IDF (NG-IDF) curves, which characterize the actual water reaching the land surface, are introduced into the design process to improve hydrologic design. The authors compared peak design flood estimates from the National Resource Conservation Service TR-55 hydrologic model driven by NG-IDF and PREC-IDF curves at 399 Snowpackmore » Telemetry (SNOTEL) stations across the western United States, all of which had at least 30 years of high-quality records. They found that about 72% of the stations in the western United States showed the potential for underdesign, for which the PREC-IDF curves underestimated peak design floods by as much as 324%. These results demonstrated the need to update the use of PREC-IDF curves to the use of NG-IDF curves for hydrologic design in snow-dominated regions.« less

Next-Generation Intensity‐Duration‐Frequency Curves for Hydrologic Design in Snow-Dominated Environments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Hongxiang; Sun, Ning; Wigmosta, Mark S.

Precipitation-based intensity-duration-frequency (PREC-IDF) curves are a standard tool used to derive design floods for hydraulic infrastructure worldwide. In snow-dominated regions where a large percentage of flood events are caused by snowmelt and rain-on-snow events, the PREC-IDF design approach can lead to substantial underestimation/overestimation of design floods and associated infrastructure. In this study, next-generation IDF (NG-IDF) curves, which characterize the actual water reaching the land surface, are introduced into the design process to improve hydrologic design. The authors compared peak design flood estimates from the National Resource Conservation Service TR-55 hydrologic model driven by NG-IDF and PREC-IDF curves at 399 Snowpackmore » Telemetry (SNOTEL) stations across the western United States, all of which had at least 30 years of high-quality records. They found that about 72% of the stations in the western United States showed the potential for underdesign, for which the PREC-IDF curves underestimated peak design floods by as much as 324%. These results demonstrated the need to update the use of PREC-IDF curves to the use of NG-IDF curves for hydrologic design in snow-dominated regions.« less

Reference Curve for the Mean Uterine Artery Pulsatility Index in Singleton Pregnancies.

PubMed

Weichert, Alexander; Hagen, Andreas; Tchirikov, Michael; Fuchs, Ilka B; Henrich, Wolfgang; Entezami, Michael

2017-05-01

Doppler sonography of the uterine artery (UA) is done to monitor pregnancies, because the detected flow patterns are useful to draw inferences about possible disorders of trophoblast invasion. Increased resistance in the UA is associated with an increased risk of preeclampsia and/or intrauterine growth restriction (IUGR) and perinatal mortality. In the absence of standardized figures, the normal ranges of the various available reference curves sometimes differ quite substantially from one another. The causes for this are differences in the flow patterns of the UA depending on the position of the pulsed Doppler gates as well as branching of the UA. Because of the discrepancies between the different reference curves and the practical problems this poses for guideline recommendations, we thought it would be useful to create our own reference curves for Doppler measurements of the UA obtained from a singleton cohort under standardized conditions. This retrospective cohort study was carried out in the Department of Obstetrics of the Charité - Universitätsmedizin Berlin, the Department for Obstetrics and Prenatal Medicine of the University Hospital Halle (Saale) and the Center for Prenatal Diagnostics and Human Genetics Kurfürstendamm 199. Available datasets from the three study locations were identified and reference curves were generated using the LMS method. Measured values were correlated with age of gestation, and a cubic model and Box-Cox power transformation (L), the median (M) and the coefficient of variation (S) were used to smooth the curves. 103 720 Doppler examinations of the UA carried out in singleton pregnancies from the 11th week of gestation (10 + 1 GW) were analyzed. The mean pulsatility index (Mean PI) showed a continuous decline over the course of pregnancy, dropping to a plateau of around 0.84 between the 23rd and 27th GW, after which it decreased again. Age of gestation, placental position, position of pulsed Doppler gates and branching of

An ROC-type measure of diagnostic accuracy when the gold standard is continuous-scale.

PubMed

Obuchowski, Nancy A

2006-02-15

ROC curves and summary measures of accuracy derived from them, such as the area under the ROC curve, have become the standard for describing and comparing the accuracy of diagnostic tests. Methods for estimating ROC curves rely on the existence of a gold standard which dichotomizes patients into disease present or absent. There are, however, many examples of diagnostic tests whose gold standards are not binary-scale, but rather continuous-scale. Unnatural dichotomization of these gold standards leads to bias and inconsistency in estimates of diagnostic accuracy. In this paper, we propose a non-parametric estimator of diagnostic test accuracy which does not require dichotomization of the gold standard. This estimator has an interpretation analogous to the area under the ROC curve. We propose a confidence interval for test accuracy and a statistical test for comparing accuracies of tests from paired designs. We compare the performance (i.e. CI coverage, type I error rate, power) of the proposed methods with several alternatives. An example is presented where the accuracies of two quick blood tests for measuring serum iron concentrations are estimated and compared.

Height velocity curves in female patients with idiopathic scoliosis.

PubMed

Chazono, Masaaki; Soshi, Sigeru; Kida, Yoshikuni; Hashimoto, Kurando; Inoue, Takeshi; Nakamura, Yousuke; Shinohara, Akira; Marumo, Keishi; Kono, Katsuki; Suzuki, Nobumasa

2012-01-01

Following identification of peak height velocity (PHV) by a recent study as a possible prognostic factor for curve progression in patients with idiopathic scoliosis (IS), the aim of this study was to investigate PHV curves in Japanese female patients with IS. The study subjects were 20 skeletally immature IS patients who were followed until maturity. The mean age and the mean pubertal status at the initial visit were 9.8 years and 24 months before menarche, respectively, with a follow-up period of 5.2 years. Height measurements were recorded at each visit, and HV was calculated as the change in height (cm) divided by the time interval (yr.) between visits of 6 to 12 months. The PHV, age at PHV (APHV), height at PHV (HPHV), and final height (FH) were determined. Patient HV curves were plotted using their HV data, and growth periods (GPs) were calculated from the curves. PHVs and GPs of study patients were compared to standard data from unaffected girls. The median values and interquartile ranges in PHV, APHV, HPHV, and FH were 8.5 cm/yr. (7.9-9.7), 11.8 yr. (11.2-12.1), 153.2 cm (150.1-155.8), and 160.1 cm (157.4-162.4), respectively. The median GP was 27 months. The PHV and GP values in IS female patients were higher and shorter than those in unaffected girls. These findings indicate that the patterns of height velocity curves in IS patients are different from those in unaffected girls, suggesting that curve progression in IS patients is associated with the magnitude of PHV and duration of GP. Recently, we have developed an HV reader to easily and quickly identify the present HV in patients with scoliosis, applicable for the clinical setting or school screening. We conclude that risk assessments of curve progression in patients with IS should include HV along with measures of skeletal maturity such as the Risser sign and/or digital skeletal age using hand X-rays.

Disease risk curves.

PubMed

Hughes, G; Burnett, F J; Havis, N D

2013-11-01

Disease risk curves are simple graphical relationships between the probability of need for treatment and evidence related to risk factors. In the context of the present article, our focus is on factors related to the occurrence of disease in crops. Risk is the probability of adverse consequences; specifically in the present context it denotes the chance that disease will reach a threshold level at which crop protection measures can be justified. This article describes disease risk curves that arise when risk is modeled as a function of more than one risk factor, and when risk is modeled as a function of a single factor (specifically the level of disease at an early disease assessment). In both cases, disease risk curves serve as calibration curves that allow the accumulated evidence related to risk to be expressed on a probability scale. When risk is modeled as a function of the level of disease at an early disease assessment, the resulting disease risk curve provides a crop loss assessment model in which the downside is denominated in terms of risk rather than in terms of yield loss.

Standard UBV Observations at the Çanakkale University Observatory (ÇUO)

NASA Astrophysics Data System (ADS)

Bakis, Hicran; Bakis, Volkan; Demircan, Osman; Budding, Edwin

2005-07-01

By using standard and comparison star observations carried out at different times of the year, at Çanakkale Onsekiz Mart University Observatory, we obtained the atmospheric extinction coefficients at the observatory. We also obtained transformation coefficients and zero-point constants for the transformation to the standard Johnson UBV system, of observations in the local system carried out with the SSP5A photometer and T40 telescope. The transmission curves and the mean wavelengths of the UBV filters as measured in the laboratory appear not much different from those of the standard Johnson system and found inside the transmission curve of the standard mean atmosphere.

On a framework for generating PoD curves assisted by numerical simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subair, S. Mohamed, E-mail: prajagopal@iitm.ac.in; Agrawal, Shweta, E-mail: prajagopal@iitm.ac.in; Balasubramaniam, Krishnan, E-mail: prajagopal@iitm.ac.in

2015-03-31

The Probability of Detection (PoD) curve method has emerged as an important tool for the assessment of the performance of NDE techniques, a topic of particular interest to the nuclear industry where inspection qualification is very important. The conventional experimental means of generating PoD curves though, can be expensive, requiring large data sets (covering defects and test conditions), and equipment and operator time. Several methods of achieving faster estimates for PoD curves using physics-based modelling have been developed to address this problem. Numerical modelling techniques are also attractive, especially given the ever-increasing computational power available to scientists today. Here wemore » develop procedures for obtaining PoD curves, assisted by numerical simulation and based on Bayesian statistics. Numerical simulations are performed using Finite Element analysis for factors that are assumed to be independent, random and normally distributed. PoD curves so generated are compared with experiments on austenitic stainless steel (SS) plates with artificially created notches. We examine issues affecting the PoD curve generation process including codes, standards, distribution of defect parameters and the choice of the noise threshold. We also study the assumption of normal distribution for signal response parameters and consider strategies for dealing with data that may be more complex or sparse to justify this. These topics are addressed and illustrated through the example case of generation of PoD curves for pulse-echo ultrasonic inspection of vertical surface-breaking cracks in SS plates.« less

On a framework for generating PoD curves assisted by numerical simulations

NASA Astrophysics Data System (ADS)

Subair, S. Mohamed; Agrawal, Shweta; Balasubramaniam, Krishnan; Rajagopal, Prabhu; Kumar, Anish; Rao, Purnachandra B.; Tamanna, Jayakumar

2015-03-01

The Probability of Detection (PoD) curve method has emerged as an important tool for the assessment of the performance of NDE techniques, a topic of particular interest to the nuclear industry where inspection qualification is very important. The conventional experimental means of generating PoD curves though, can be expensive, requiring large data sets (covering defects and test conditions), and equipment and operator time. Several methods of achieving faster estimates for PoD curves using physics-based modelling have been developed to address this problem. Numerical modelling techniques are also attractive, especially given the ever-increasing computational power available to scientists today. Here we develop procedures for obtaining PoD curves, assisted by numerical simulation and based on Bayesian statistics. Numerical simulations are performed using Finite Element analysis for factors that are assumed to be independent, random and normally distributed. PoD curves so generated are compared with experiments on austenitic stainless steel (SS) plates with artificially created notches. We examine issues affecting the PoD curve generation process including codes, standards, distribution of defect parameters and the choice of the noise threshold. We also study the assumption of normal distribution for signal response parameters and consider strategies for dealing with data that may be more complex or sparse to justify this. These topics are addressed and illustrated through the example case of generation of PoD curves for pulse-echo ultrasonic inspection of vertical surface-breaking cracks in SS plates.

Tornado-Shaped Curves

ERIC Educational Resources Information Center

Martínez, Sol Sáez; de la Rosa, Félix Martínez; Rojas, Sergio

2017-01-01

In Advanced Calculus, our students wonder if it is possible to graphically represent a tornado by means of a three-dimensional curve. In this paper, we show it is possible by providing the parametric equations of such tornado-shaped curves.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

PubMed

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

PubMed Central

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

Constraining brane tension using rotation curves of galaxies

NASA Astrophysics Data System (ADS)

García-Aspeitia, Miguel A.; Rodríguez-Meza, Mario A.

2018-04-01

We present in this work a study of brane theory phenomenology focusing on the brane tension parameter, which is the main observable of the theory. We show the modifications steaming from the presence of branes in the rotation curves of spiral galaxies for three well known dark matter density profiles: Pseudo isothermal, Navarro-Frenk-White and Burkert dark matter density profiles. We estimate the brane tension parameter using a sample of high resolution observed rotation curves of low surface brightness spiral galaxies and a synthetic rotation curve for the three density profiles. Also, the fittings using the brane theory model of the rotation curves are compared with standard Newtonian models. We found that Navarro-Frenk-White model prefers lower values of the brane tension parameter, on the average λ ∼ 0.73 × 10‑3eV4, therefore showing clear brane effects. Burkert case does prefer higher values of the tension parameter, on the average λ ∼ 0.93 eV4 ‑ 46 eV4, i.e., negligible brane effects. Whereas pseudo isothermal is an intermediate case. Due to the low densities found in the galactic medium it is almost impossible to find evidence of the presence of extra dimensions. In this context, we found that our results show weaker bounds to the brane tension values in comparison with other bounds found previously, as the lower value found for dwarf stars composed of a polytropic equation of state, λ ≈ 104 MeV4.

Data in support of qPCR primer design and verification in a Pink1 -/- rat model of Parkinson disease.

PubMed

Kelm-Nelson, Cynthia A; Stevenson, Sharon A; Ciucci, Michelle R

2016-09-01

Datasets provided in this article represent the Rattus norvegicus primer design and verification used in Pink1 -/- and wildtype Long Evans brain tissue. Accessible tables include relevant information, accession numbers, sequences, temperatures and product length, describing primer design specific to the transcript amplification use. Additionally, results of Sanger sequencing of qPCR reaction products (FASTA aligned sequences) are presented for genes of interest. Results and further interpretation and discussion can be found in the original research article "Atp13a2 expression in the periaqueductal gray is decreased in the Pink1 -/- rat model of Parkinson disease" [1].

In-Vehicle Dynamic Curve-Speed Warnings at High-Risk Rural Curves

DOT National Transportation Integrated Search

2018-03-01

Lane-departure crashes at horizontal curves represent a significant portion of fatal crashes on rural Minnesota roads. Because of this, solutions are needed to aid drivers in identifying upcoming curves and inform them of a safe speed at which they s...

Are precipitation-based intensity-duration-frequency curves appropriate for cost effective and resilient infrastructure design in snow-dominated regions? Next-generation curves with inclusion of rain-on-snow events

NASA Astrophysics Data System (ADS)

Yan, H.; Sun, N.; Wigmosta, M. S.; Hou, Z.

2017-12-01

There is a renewed focus on the design of infrastructure resilient to extreme hydrometeorological events. While precipitation-based intensity-duration-frequency (IDF) curves are commonly used as part of infrastructure design, a large percentage of peak runoff events in the snow-dominated regions are caused by snowmelt, particularly during rain-on-snow (ROS) events. In this study, we examined next-generation IDF (NG-IDF) curves with inclusion of snowmelt and ROS events to improve infrastructure design in snow-dominated regions. We compared NG-IDF curves to standard precipitation-based IDF curves for estimates of extreme events at 377 Snowpack Telemetry (SNOTEL) stations across the western United States with at least 30 years of high quality record. We found 38% of the stations were subject to under-design, many with significant underestimation of 100-year extreme events, where the precipitation-based IDF curves can underestimate water potentially available for runoff by as much as 121% due to snowmelt and ROS events. The regions with the greatest potential for under-design were in the Pacific Northwest, the Sierra Nevada, and the Middle and Southern Rockies. We also found the potential for over-design at 27% of the stations, primarily in the Middle Rockies and Arizona mountains. These results demonstrate the need to consider snow processes in development of IDF curves for engineering design procedures in snow-dominated regions.

A System of Standard Atmospheres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Charles, B.N.

1954-04-06

This report included a proposed new set of Standard Atmospheres mainly to help with the construction and use of the baro tables for the Mk 6 weapon. Temperature-height curves over the world can assume an infinte number of configurations.

DEVELOPMENT OF DATA QUALITY OBJECTIVES AND USE OF TWO VARIATIONS OF GENETICALLY-MODIFIED STREPTOCOCCUS GORDONIL AS LYSIS CONTROLS IN A QPCR ASSAY FOR ASSESSING SANITARY QUALITY OF WATER

EPA Science Inventory

Joseph B. James and Fred J. Genthner

United States Environmental Protection Agency, Gulf Breeze, FL

Background: Methods using rapid cycle, real-time, quantitative (QPCR) are being developed for detecting and quantifying Enterococcus spp. as well as other aquatic b...

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

PubMed Central

Banting, Graham S.; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia

2016-01-01

ABSTRACT Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari. Campylobacters in raw sewage were present at ∼102/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease

Estimation of Uncertainties in Stage-Discharge Curve for an Experimental Himalayan Watershed

NASA Astrophysics Data System (ADS)

Kumar, V.; Sen, S.

2016-12-01

Various water resource projects developed on rivers originating from the Himalayan region, the "Water Tower of Asia", plays an important role on downstream development. Flow measurements at the desired river site are very critical for river engineers and hydrologists for water resources planning and management, flood forecasting, reservoir operation and flood inundation studies. However, an accurate discharge assessment of these mountainous rivers is costly, tedious and frequently dangerous to operators during flood events. Currently, in India, discharge estimation is linked to stage-discharge relationship known as rating curve. This relationship would be affected by a high degree of uncertainty. Estimating the uncertainty of rating curve remains a relevant challenge because it is not easy to parameterize. Main source of rating curve uncertainty are errors because of incorrect discharge measurement, variation in hydraulic conditions and depth measurement. In this study our objective is to obtain best parameters of rating curve that fit the limited record of observations and to estimate uncertainties at different depth obtained from rating curve. The rating curve parameters of standard power law are estimated for three different streams of Aglar watershed located in lesser Himalayas by maximum-likelihood estimator. Quantification of uncertainties in the developed rating curves is obtained from the estimate of variances and covariances of the rating curve parameters. Results showed that the uncertainties varied with catchment behavior with error varies between 0.006-1.831 m3/s. Discharge uncertainty in the Aglar watershed streams significantly depend on the extent of extrapolation outside the range of observed water levels. Extrapolation analysis confirmed that more than 15% for maximum discharges and 5% for minimum discharges are not strongly recommended for these mountainous gauging sites.

Fingerprinting breakthrough curves in soils

NASA Astrophysics Data System (ADS)

Koestel, J. K.

2017-12-01

Conservative solute transport through soil is predominantly modeled using a few standard solute transport models like the convection dispersion equation or the mobile-immobile model. The adequacy of these models is seldom investigated in detail as it would require knowledge on the 3-D spatio-temporal evolution of the solute plume that is normally not available. Instead, shape-measures of breakthrough curves (BTCs) such as the apparent dispersivity and the relative 5%-arrival time may be used to fingerprint breakthrough curves as well as forward solutions of solute transport models. In this fashion the similarity of features from measured and modeled BTC data becomes quantifiable. In this study I am presenting a new set of shape-measures that characterize the log-log tailings of BTC. I am using the new shape measures alongside with more established ones to map the features of BTCs obtained forward models of the convective dispersive equation, log-normal and Gamma transfer functions, the mobile-immobile model and the continuous time random walk model with respect to their input parameters. In a second step, I am comparing corresponding shape-measures for 206 measured BTCs extracted from peer-reviewed literature. Preliminary results show that power-law tailings are very common in BTCs from soil samples and that BTC features that are exclusive to a mobile-immobile type solute transport process are very rarely found.

Growth standard charts for monitoring bodyweight in dogs of different sizes

PubMed Central

Salt, Carina; Morris, Penelope J.; Wilson, Derek; Lund, Elizabeth M.; Cole, Tim J.; Butterwick, Richard F.

2017-01-01

Limited information is available on what constitutes optimal growth in dogs. The primary aim of this study was to develop evidence-based growth standards for dogs, using retrospective analysis of bodyweight and age data from >6 million young dogs attending a large corporate network of primary care veterinary hospitals across the USA. Electronic medical records were used to generate bodyweight data from immature client-owned dogs, that were healthy and had remained in ideal body condition throughout the first 3 years of life. Growth centile curves were constructed using Generalised Additive Models for Location, Shape and Scale. Curves were displayed graphically as centile charts covering the age range 12 weeks to 2 years. Over 100 growth charts were modelled, specific to different combinations of breed, sex and neuter status. Neutering before 37 weeks was associated with a slight upward shift in growth trajectory, whilst neutering after 37 weeks was associated with a slight downward shift in growth trajectory. However, these shifts were small in comparison to inter-individual variability amongst dogs, suggesting that separate curves for neutered dogs were not needed. Five bodyweight categories were created to cover breeds up to 40kg, using both visual assessment and hierarchical cluster analysis of breed-specific growth curves. For 20/24 of the individual breed centile curves, agreement with curves for the corresponding bodyweight categories was good. For the remaining 4 breed curves, occasional deviation across centile lines was observed, but overall agreement was acceptable. This suggested that growth could be described using size categories rather than requiring curves for specific breeds. In the current study, a series of evidence-based growth standards have been developed to facilitate charting of bodyweight in healthy dogs. Additional studies are required to validate these standards and create a clinical tool for growth monitoring in pet dogs. PMID:28873413

Growth standard charts for monitoring bodyweight in dogs of different sizes.

PubMed

Salt, Carina; Morris, Penelope J; German, Alexander J; Wilson, Derek; Lund, Elizabeth M; Cole, Tim J; Butterwick, Richard F

2017-01-01

Limited information is available on what constitutes optimal growth in dogs. The primary aim of this study was to develop evidence-based growth standards for dogs, using retrospective analysis of bodyweight and age data from >6 million young dogs attending a large corporate network of primary care veterinary hospitals across the USA. Electronic medical records were used to generate bodyweight data from immature client-owned dogs, that were healthy and had remained in ideal body condition throughout the first 3 years of life. Growth centile curves were constructed using Generalised Additive Models for Location, Shape and Scale. Curves were displayed graphically as centile charts covering the age range 12 weeks to 2 years. Over 100 growth charts were modelled, specific to different combinations of breed, sex and neuter status. Neutering before 37 weeks was associated with a slight upward shift in growth trajectory, whilst neutering after 37 weeks was associated with a slight downward shift in growth trajectory. However, these shifts were small in comparison to inter-individual variability amongst dogs, suggesting that separate curves for neutered dogs were not needed. Five bodyweight categories were created to cover breeds up to 40kg, using both visual assessment and hierarchical cluster analysis of breed-specific growth curves. For 20/24 of the individual breed centile curves, agreement with curves for the corresponding bodyweight categories was good. For the remaining 4 breed curves, occasional deviation across centile lines was observed, but overall agreement was acceptable. This suggested that growth could be described using size categories rather than requiring curves for specific breeds. In the current study, a series of evidence-based growth standards have been developed to facilitate charting of bodyweight in healthy dogs. Additional studies are required to validate these standards and create a clinical tool for growth monitoring in pet dogs.

Site-index curves for young-growth ponderosa pine in northern Arizona

Treesearch

Charles O. Minor

1964-01-01

The productive capacity or site quality of an area enters into nearly every phase of forest management from regeneration to final harvest. No standards or measures of site quality have been developed specifically for ponderosa pine in the Southwest, which handicaps the forest manager. The major objective of the present study was to develop the basic site-index curves...

Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

PubMed

Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

2015-08-01

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

CMV viral load measurements in whole blood and plasma--which is best following renal transplantation?

PubMed

Barrett-Muir, W; Breuer, J; Millar, C; Thomas, J; Jeffries, D; Yaqoob, M; Aitken, C

2000-07-15

Quantitative commercial assays for cytomegalovirus (CMV) detection have recently been developed. Their role in the management of patients after transplantation needs to be evaluated. Widespread use of these assays will allow for comparison of results between centers and meaningful interpretation of the significance of viral load measurements. Sequential samples from 52 patients after renal transplantation were tested in the murex hybrid capture assay (HCA) and the Roche Amplicor CMV DNA assay (QPCR) and correlated with the development of CMV disease. A comparison of viral loads in plasma and whole blood was also made. Both assays were sensitive and detected all cases of CMV disease. The specificity and positive predictive value increased from 0.34 and 0.36 to 0.85 and 0.96 for the HCA and 0.37, 0.37 to 0.72 and 0.63 for the QPCR following a receiver operator curve analysis. Higher viral loads were measured using the HCA compared to the QPCR. Response to ganciclovir was associated with a greater than 80% reduction in viral load by HCA or greater than 70% using the QPCR. Both assays were highly sensitive. By using a receiver operator curve analysis a cutoff viral load can be determined which maximizes the clinical utility of these assays.

Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin.

PubMed

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

Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

PubMed

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

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

PubMed

Glenney, Gavin W; Barbash, Patricia A; Coll, John A

2016-03-01

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

Standard Errors and Confidence Intervals from Bootstrapping for Ramsay-Curve Item Response Theory Model Item Parameters

ERIC Educational Resources Information Center

Gu, Fei; Skorupski, William P.; Hoyle, Larry; Kingston, Neal M.

2011-01-01

Ramsay-curve item response theory (RC-IRT) is a nonparametric procedure that estimates the latent trait using splines, and no distributional assumption about the latent trait is required. For item parameters of the two-parameter logistic (2-PL), three-parameter logistic (3-PL), and polytomous IRT models, RC-IRT can provide more accurate estimates…

Percentile Curves for Anthropometric Measures for Canadian Children and Youth.

PubMed

Kuhle, Stefan; Maguire, Bryan; Ata, Nicole; Hamilton, David

2015-01-01

Body mass index (BMI) is commonly used to assess a child's weight status but it does not provide information about the distribution of body fat. Since the disease risks associated with obesity are related to the amount and distribution of body fat, measures that assess visceral or subcutaneous fat, such as waist circumference (WC), waist-to-height ratio (WHtR), or skinfolds thickness may be more suitable. The objective of this study was to develop percentile curves for BMI, WC, WHtR, and sum of 5 skinfolds (SF5) in a representative sample of Canadian children and youth. The analysis used data from 4115 children and adolescents between 6 and 19 years of age that participated in the Canadian Health Measures Survey Cycles 1 (2007/2009) and 2 (2009/2011). BMI, WC, WHtR, and SF5 were measured using standardized procedures. Age- and sex-specific centiles were calculated using the LMS method and the percentiles that intersect the adult cutpoints for BMI, WC, and WHtR at age 18 years were determined. Percentile curves for all measures showed an upward shift compared to curves from the pre-obesity epidemic era. The adult cutoffs for overweight and obesity corresponded to the 72nd and 91st percentile, respectively, for both sexes. The current study has presented for the first time percentile curves for BMI, WC, WHtR, and SF5 in a representative sample of Canadian children and youth. The percentile curves presented are meant to be descriptive rather than prescriptive as associations with cardiovascular disease markers or outcomes were not assessed.

Testing modified gravity at large distances with the HI Nearby Galaxy Survey's rotation curves

NASA Astrophysics Data System (ADS)

Mastache, Jorge; Cervantes-Cota, Jorge L.; de la Macorra, Axel

2013-03-01

Recently a new—quantum motivated—theory of gravity has been proposed that modifies the standard Newtonian potential at large distances when spherical symmetry is considered. Accordingly, Newtonian gravity is altered by adding an extra Rindler acceleration term that has to be phenomenologically determined. Here we consider a standard and a power-law generalization of the Rindler modified Newtonian potential. The new terms in the gravitational potential are hypothesized to play the role of dark matter in galaxies. Our galactic model includes the mass of the integrated gas, and stars for which we consider three stellar mass functions (Kroupa, diet-Salpeter, and free mass model). We test this idea by fitting rotation curves of seventeen low surface brightness galaxies from the HI Nearby Galaxy Survey (THINGS). We find that the Rindler parameters do not perform a suitable fit to the rotation curves in comparison to standard dark matter profiles (Navarro-Frenk-White and Burkert) and, in addition, the computed parameters of the Rindler gravity show a high spread, posing the model as a nonacceptable alternative to dark matter.

Comparing rapid and culture indicator bacteria methods at inland lake beaches

USGS Publications Warehouse

Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Kephart, Christopher M.

2013-01-01

A rapid method, quantitative polymerase chain reaction (qPCR), for quantifying indicator bacteria in recreational waters is desirable for public health protection. We report that replacing current Escherichia coli standards with new US Environmental Protection Agency beach action values (BAVs) for enterococci by culture or qPCR may result in more advisories being posted at inland recreational lakes. In this study, concentrations of E. coli and enterococci by culture methods were compared to concentrations of Enterococcus spp. by qPCR at 3 inland lake beaches in Ohio. The E. coli and enterococci culture results were significantly related at all beaches; however, the relations between culture results and Enterococcus spp. qPCR results were not always significant and differed among beaches. All the qPCR results exceeded the new BAV for Enterococcus spp. by qPCR, whereas only 23.7% of culture results for E. coli and 79% of culture results for enterococci exceeded the current standard for E. coli or BAV for enterococci.

Analysis of variation in calibration curves for Kodak XV radiographic film using model-based parameters.

PubMed

Hsu, Shu-Hui; Kulasekere, Ravi; Roberson, Peter L

2010-08-05

Film calibration is time-consuming work when dose accuracy is essential while working in a range of photon scatter environments. This study uses the single-target single-hit model of film response to fit the calibration curves as a function of calibration method, processor condition, field size and depth. Kodak XV film was irradiated perpendicular to the beam axis in a solid water phantom. Standard calibration films (one dose point per film) were irradiated at 90 cm source-to-surface distance (SSD) for various doses (16-128 cGy), depths (0.2, 0.5, 1.5, 5, 10 cm) and field sizes (5 × 5, 10 × 10 and 20 × 20 cm²). The 8-field calibration method (eight dose points per film) was used as a reference for each experiment, taken at 95 cm SSD and 5 cm depth. The delivered doses were measured using an Attix parallel plate chamber for improved accuracy of dose estimation in the buildup region. Three fitting methods with one to three dose points per calibration curve were investigated for the field sizes of 5 × 5, 10 × 10 and 20 × 20 cm². The inter-day variation of model parameters (background, saturation and slope) were 1.8%, 5.7%, and 7.7% (1 σ) using the 8-field method. The saturation parameter ratio of standard to 8-field curves was 1.083 ± 0.005. The slope parameter ratio of standard to 8-field curves ranged from 0.99 to 1.05, depending on field size and depth. The slope parameter ratio decreases with increasing depth below 0.5 cm for the three field sizes. It increases with increasing depths above 0.5 cm. A calibration curve with one to three dose points fitted with the model is possible with 2% accuracy in film dosimetry for various irradiation conditions. The proposed fitting methods may reduce workload while providing energy dependence correction in radiographic film dosimetry. This study is limited to radiographic XV film with a Lumisys scanner.

NEXT Performance Curve Analysis and Validation

NASA Technical Reports Server (NTRS)

Saripalli, Pratik; Cardiff, Eric; Englander, Jacob

2016-01-01

Performance curves of the NEXT thruster are highly important in determining the thruster's ability in performing towards mission-specific goals. New performance curves are proposed and examined here. The Evolutionary Mission Trajectory Generator (EMTG) is used to verify variations in mission solutions based on both available thruster curves and the new curves generated. Furthermore, variations in BOL and EOL curves are also examined. Mission design results shown here validate the use of EMTG and the new performance curves.

Motor development curve from 0 to 12 months in infants born preterm.

PubMed

Kayenne Martins Roberto Formiga, Cibelle; Linhares, Maria Beatriz Martins

2011-03-01

To trace a reference curve for motor development from birth up to 12 months of corrected chronological age in infants born preterm and low birth weight. This is a cross-sectional study with a sample of 308 preterm infants (53% boys) weighing < 2500 g at birth. The Alberta Infant Motor Scale (AIMS) was used for motor development assessment. Comparing the motor performance of preterm infants with infants from a standardized sample on the AIMS, it was found that, except for the age group of the newborn, preterm infants showed lower motor development scores in comparison with the AIMS normative sample in all age groups between 1 and 12 months. The curve of motor development showed a continuous increase in the number of motor skills of preterm infants during their first 12 months of age. However, the average of motor acquisitions of preterm infants showed a nonlinear pattern with a standard indicator of stabilization between 8 and 10 months of age. Preterm infants, 1-12 months of age, showed motor development AIMS scores lower than the standards established in the normative sample. The findings may contribute as norm-reference for assessing the motor development of preterm infants in follow-up programmes in developing countries. © 2010 The Author(s)/Acta Paediatrica © 2010 Foundation Acta Paediatrica.

Experimental determination of the yield stress curve of the scotch pine wood materials

NASA Astrophysics Data System (ADS)

Günay, Ezgi; Aygün, Cevdet; Kaya, Şükrü Tayfun

2013-12-01

Yield stress curve is determined for the pine wood specimens by conducting a series of tests. In this work, pinewood is modeled as a composite material with transversely isotropic fibers. Annual rings (wood grain) of the wood specimens are taken as the major fiber directions with which the strain gauge directions are aligned. For this purpose, three types of tests are arranged. These are tensile, compression and torsion loading tests. All of the tests are categorized with respect to fiber orientations and their corresponding loading conditions. Each test within these categories is conducted separately. Tensile and compression tests are conducted in accordance with standards of Turkish Standards Institution (TSE) whereas torsion tests are conducted in accordance with Standards Australia. Specimens are machined from woods of Scotch pine which is widely used in boat building industries and in other structural engineering applications. It is determined that this species behaves more flexibly than the others. Strain gauges are installed on the specimen surfaces in such a way that loading measurements are performed along directions either parallel or perpendicular to the fiber directions. During the test and analysis phase of yield stress curve, orientation of strain gauge directions with respect to fiber directions are taken into account. The diagrams of the normal stress vs. normal strain or the shear stress vs. shear strain are plotted for each test. In each plot, the yield stress is determined by selecting the point on the diagram, the tangent of which is having a slope of 5% less than the slope of the elastic portion of the diagram. The geometric locus of these selected points constitutes a single yield stress curve on σ1-σ2 principal plane. The resulting yield stress curve is plotted as an approximate ellipse which resembles Tsai-Hill failure criterion. The results attained in this work, compare well with the results which are readily available in the literature.

Quantifying the safety effects of horizontal curves on two-way, two-lane rural roads.

PubMed

Gooch, Jeffrey P; Gayah, Vikash V; Donnell, Eric T

2016-07-01

The objective of this study is to quantify the safety performance of horizontal curves on two-way, two-lane rural roads relative to tangent segments. Past research is limited by small samples sizes, outdated statistical evaluation methods, and unreported standard errors. This study overcomes these drawbacks by using the propensity scores-potential outcomes framework. The impact of adjacent curves on horizontal curve safety is also explored using a cross-sectional regression model of only horizontal curves. The models estimated in the present study used eight years of crash data (2005-2012) obtained from over 10,000 miles of state-owned two-lane rural roads in Pennsylvania. These data included information on roadway geometry (e.g., horizontal curvature, lane width, and shoulder width), traffic volume, roadside hazard rating, and the presence of various low-cost safety countermeasures (e.g., centerline and shoulder rumble strips, curve and intersection warning pavement markings, and aggressive driving pavement dots). Crash prediction is performed by means of mixed effects negative binomial regression using the explanatory variables noted previously, as well as attributes of adjacent horizontal curves. The results indicate that both the presence of a horizontal curve and its degree of curvature must be considered when predicting the frequency of total crashes on horizontal curves. Both are associated with an increase in crash frequency, which is consistent with previous findings in the literature. Mixed effects negative binomial regression models for total crash frequency on horizontal curves indicate that the distance to adjacent curves is not statistically significant. However, the degree of curvature of adjacent curves in close proximity (within 0.75 miles) was found to be statistically significant and negatively correlated with crash frequency on the subject curve. This is logical, as drivers exiting a sharp curve are likely to be driving slower and with more

Proficiency Standards and Cut-Scores for Language Proficiency Tests.

ERIC Educational Resources Information Center

Moy, Raymond H.

The problem of standard setting on language proficiency tests is often approached by the use of norms derived from the group being tested, a process commonly known as "grading on the curve." One particular problem with this ad hoc method of standard setting is that it will usually result in a fluctuating standard dependent on the particular group…

Application of standard addition for the determination of carboxypeptidase activity in Actinomucor elegans bran koji.

PubMed

Fu, J; Li, L; Yang, X Q; Zhu, M J

2011-01-01

Leucine carboxypeptidase (EC 3.4.16) activity in Actinomucor elegans bran koji was investigated via absorbance at 507 nm after stained by Cd-nihydrin solution, with calibration curve A, which was made by a set of known concentration standard leucine, calibration B, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations inactive crude enzyme extract, and calibration C, which was made by three sets of known concentration standard leucine solutions with the addition of three concentrations crude enzyme extract. The results indicated that application of pure amino acid standard curve was not a suitable way to determine carboxypeptidase in complicate mixture, and it probably led to overestimated carboxypeptidase activity. It was found that addition of crude exact into pure amino acid standard curve had a significant difference from pure amino acid standard curve method (p < 0.05). There was no significant enzyme activity difference (p > 0.05) between addition of active crude exact and addition of inactive crude kind, when the proper dilute multiple was used. It was concluded that the addition of crude enzyme extract to the calibration was needed to eliminate the interference of free amino acids and related compounds presented in crude enzyme extract.

Nonlinear Curve-Fitting Program

NASA Technical Reports Server (NTRS)

Everhart, Joel L.; Badavi, Forooz F.

1989-01-01

Nonlinear optimization algorithm helps in finding best-fit curve. Nonlinear Curve Fitting Program, NLINEAR, interactive curve-fitting routine based on description of quadratic expansion of X(sup 2) statistic. Utilizes nonlinear optimization algorithm calculating best statistically weighted values of parameters of fitting function and X(sup 2) minimized. Provides user with such statistical information as goodness of fit and estimated values of parameters producing highest degree of correlation between experimental data and mathematical model. Written in FORTRAN 77.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using FTIR-ATR Spectroscopy to Teach the Internal Standard Method

ERIC Educational Resources Information Center

Bellamy, Michael K.

2010-01-01

The internal standard method is widely applied in quantitative analyses. However, most analytical chemistry textbooks either omit this topic or only provide examples of a single-point internal standardization. An experiment designed to teach students how to prepare an internal standard calibration curve is described. The experiment is a modified…

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

PubMed

Linck, Holger; Krüger, Erika; Reineke, Annette

2017-01-01

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.

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

PubMed Central

Krüger, Erika; Reineke, Annette

2017-01-01

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. PMID:28545043

An alternative approach to the Army Physical Fitness Test two-mile run using critical velocity and isoperformance curves.

PubMed

Fukuda, David H; Smith, Abbie E; Kendall, Kristina L; Cramer, Joel T; Stout, Jeffrey R

2012-02-01

The purpose of this study was to evaluate the use of critical velocity (CV) and isoperformance curves as an alternative to the Army Physical Fitness Test (APFT) two-mile running test. Seventy-eight men and women (mean +/- SE; age: 22.1 +/- 0.34 years; VO2(MAX): 46.1 +/- 0.82 mL/kg/min) volunteered to participate in this study. A VO2(MAX) test and four treadmill running bouts to exhaustion at varying intensities were completed. The relationship between total distance and time-to-exhaustion was tracked for each exhaustive run to determine CV and anaerobic running capacity. A VO2(MAX) prediction equation (Coefficient of determination: 0.805; Standard error of the estimate: 3.2377 mL/kg/min) was developed using these variables. Isoperformance curves were constructed for men and women to correspond with two-mile run times from APFT standards. Individual CV and anaerobic running capacity values were plotted and compared to isoperformance curves for APFT 2-mile run scores. Fifty-four individuals were determined to receive passing scores from this assessment. Physiological profiles identified from this procedure can be used to assess specific aerobic or anaerobic training needs. With the use of time-to-exhaustion as opposed to a time-trial format used in the two-mile run test, pacing strategies may be limited. The combination of variables from the CV test and isoperformance curves provides an alternative to standardized time-trial testing.

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

PubMed

Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

2017-10-02

Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Preliminary Investigation of Curved Liner Sample in the NASA LaRC Curved Duct Test Rig

NASA Technical Reports Server (NTRS)

Gerhold, Carl H.; Jones, Michael G.; Brown, Martha C.

2007-01-01

This viewgraph presentation reviews the preliminary investigation of the curved liner sample in the NASA LaRC Curved Duct Test Rig (CDTR). It reviews the purpose of the Curved Duct Test Rig. Its purpose is to develop capability to investigate acoustic and aerodynamic properties in ducts. It has several features to accomplish that purpose: (1) Large scale (2) Flow rate to M = 0.275 (3) Higher order mode control (4) Curved flow path (5) Adaptable test section (6) Flexible test configurations. The liner has minimal effect on turbulence or boundary layer growth in duct. The curved duct sample attenuation is affected by mode scattering. In conclusion, the CDTR is valid tool for aerodynamic and acoustic evaluation of duct treatment

Percentile Curves for Anthropometric Measures for Canadian Children and Youth

PubMed Central

Kuhle, Stefan; Maguire, Bryan; Ata, Nicole; Hamilton, David

2015-01-01

Body mass index (BMI) is commonly used to assess a child's weight status but it does not provide information about the distribution of body fat. Since the disease risks associated with obesity are related to the amount and distribution of body fat, measures that assess visceral or subcutaneous fat, such as waist circumference (WC), waist-to-height ratio (WHtR), or skinfolds thickness may be more suitable. The objective of this study was to develop percentile curves for BMI, WC, WHtR, and sum of 5 skinfolds (SF5) in a representative sample of Canadian children and youth. The analysis used data from 4115 children and adolescents between 6 and 19 years of age that participated in the Canadian Health Measures Survey Cycles 1 (2007/2009) and 2 (2009/2011). BMI, WC, WHtR, and SF5 were measured using standardized procedures. Age- and sex-specific centiles were calculated using the LMS method and the percentiles that intersect the adult cutpoints for BMI, WC, and WHtR at age 18 years were determined. Percentile curves for all measures showed an upward shift compared to curves from the pre-obesity epidemic era. The adult cutoffs for overweight and obesity corresponded to the 72nd and 91st percentile, respectively, for both sexes. The current study has presented for the first time percentile curves for BMI, WC, WHtR, and SF5 in a representative sample of Canadian children and youth. The percentile curves presented are meant to be descriptive rather than prescriptive as associations with cardiovascular disease markers or outcomes were not assessed. PMID:26176769

A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater.

PubMed

Jin, Yang Oh; Mattes, Timothy E

2010-12-01

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.

Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves.

PubMed

Sprouffske, Kathleen; Wagner, Andreas

2016-04-19

Plate readers can measure the growth curves of many microbial strains in a high-throughput fashion. The hundreds of absorbance readings collected simultaneously for hundreds of samples create technical hurdles for data analysis. Growthcurver summarizes the growth characteristics of microbial growth curve experiments conducted in a plate reader. The data are fitted to a standard form of the logistic equation, and the parameters have clear interpretations on population-level characteristics, like doubling time, carrying capacity, and growth rate. Growthcurver is an easy-to-use R package available for installation from the Comprehensive R Archive Network (CRAN). The source code is available under the GNU General Public License and can be obtained from Github (Sprouffske K, Growthcurver sourcecode, 2016).

Identification of Lactobacillus delbrueckii and Streptococcus thermophilus Strains Present in Artisanal Raw Cow Milk Cheese Using Real-time PCR and Classic Plate Count Methods.

PubMed

Stachelska, Milena A

2017-12-04

The aim of this paper was to detect Lactobacillus delbrueckii and Streptococcus thermophilus using real-time quantitative PCR assay in 7-day ripening cheese produced from unpasteurised milk. Real-time quantitative PCR assays were designed to identify and enumerate the chosen species of lactic acid bacteria (LAB) in ripened cheese. The results of molecular quantification and classic bacterial enumeration showed a high level of similarity proving that DNA extraction was carried out in a proper way and that genomic DNA solutions were free of PCR inhibitors. These methods revealed the presence of L. delbrueckii and S. thermophilus. The real-time PCR enabled quantification with a detection of 101-103 CFU/g of product. qPCR-standard curves were linear over seven log units down to 101 copies per reaction; efficiencies ranged from 77.9% to 93.6%. Cheese samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method provided faster and species specific identification of two dairy LAB and yielded comparable quantitative results.

Using quasars as standard clocks for measuring cosmological redshift.

PubMed

Dai, De-Chang; Starkman, Glenn D; Stojkovic, Branislav; Stojkovic, Dejan; Weltman, Amanda

2012-06-08

We report hitherto unnoticed patterns in quasar light curves. We characterize segments of the quasar's light curves with the slopes of the straight lines fit through them. These slopes appear to be directly related to the quasars' redshifts. Alternatively, using only global shifts in time and flux, we are able to find significant overlaps between the light curves of different pairs of quasars by fitting the ratio of their redshifts. We are then able to reliably determine the redshift of one quasar from another. This implies that one can use quasars as standard clocks, as we explicitly demonstrate by constructing two independent methods of finding the redshift of a quasar from its light curve.

Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

PubMed

Gregory, T Ryan; Nathwani, Paula; Bonnett, Tiffany R; Huber, Dezene P W

2013-09-01

A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase

PubMed Central

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. PMID:29615986

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase.

PubMed

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.

Correlation of crAssphage-based qPCR markers with culturable and molecular indicators of human fecal pollution in an impacted urban watershed.

PubMed

Stachler, Elyse; Akyon, Benay; Aquino de Carvalho, Nathalia; Ference, Christian; Bibby, Kyle

2018-06-06

Environmental waters are monitored for fecal pollution to protect public health. Many previously developed human-specific fecal pollution indicators lack adequate sensitivity to be reliably detected in environmental waters or do not correlate well with viral pathogens. Recently, two novel human sewage-associated source tracking qPCR markers were developed based on the bacteriophage crAssphage, CPQ_056 and CPQ_064. These assays are highly human specific, abundant in sewage, and are viral-based, suggesting great promise for environmental application as human fecal pollution indicators. A 30-day sampling study was conducted in an urban stream impacted by combined sewer overflows to evaluate the crAssphage markers' performance in an environmental system. The crAssphage markers were present at concentrations of 4.02-6.04 log10 copies/100 mL throughout the study period, indicating their high abundance and ease of detection in polluted environmental waters. In addition, the crAssphage assays were correlated with rain events, molecular markers for human polyomavirus and HF183, as well as culturable E. coli, enterococci, and somatic coliphage. The CPQ_064 assay correlated strongly to a greater number of biological indicators than the CPQ_056 assay. This study is the first to evaluate both crAssphage qPCR assays in an extended environmental application of crAssphage markers for monitoring of environmental waters. It is also the first study to compare crAssphage marker concentration with other viral-based indicators.

The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel.

PubMed

Ohad, Shoshanit; Ben-Dor, Shifra; Prilusky, Jaime; Kravitz, Valeria; Dassa, Bareket; Chalifa-Caspi, Vered; Kashi, Yechezkel; Rorman, Efrat

2016-01-01

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 quantitative polymerase chain reaction (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.

Titration Curves: Fact and Fiction.

ERIC Educational Resources Information Center

Chamberlain, John

1997-01-01

Discusses ways in which datalogging equipment can enable titration curves to be measured accurately and how computing power can be used to predict the shape of curves. Highlights include sources of error, use of spreadsheets to generate titration curves, titration of a weak acid with a strong alkali, dibasic acids, weak acid and weak base, and…

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

PubMed Central

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

Inverse Diffusion Curves Using Shape Optimization.

PubMed

Zhao, Shuang; Durand, Fredo; Zheng, Changxi

2018-07-01

The inverse diffusion curve problem focuses on automatic creation of diffusion curve images that resemble user provided color fields. This problem is challenging since the 1D curves have a nonlinear and global impact on resulting color fields via a partial differential equation (PDE). We introduce a new approach complementary to previous methods by optimizing curve geometry. In particular, we propose a novel iterative algorithm based on the theory of shape derivatives. The resulting diffusion curves are clean and well-shaped, and the final image closely approximates the input. Our method provides a user-controlled parameter to regularize curve complexity, and generalizes to handle input color fields represented in a variety of formats.

[Customized and non-customized French intrauterine growth curves. II - Comparison with existing curves and benefits of customization].

PubMed

Ego, A; Prunet, C; Blondel, B; Kaminski, M; Goffinet, F; Zeitlin, J

2016-02-01

Our aim is to compare the new French EPOPé intrauterine growth curves, developed to address the guidelines 2013 of the French College of Obstetricians and Gynecologists, with reference curves currently used in France, and to evaluate the consequences of their adjustment for fetal sex and maternal characteristics. Eight intrauterine and birthweight curves, used in France were compared to the EPOPé curves using data from the French Perinatal Survey 2010. The influence of adjustment on the rate of SGA births and the characteristics of these births was analysed. Due to their birthweight values and distribution, the selected intrauterine curves are less suitable for births in France than the new curves. Birthweight curves led to low rates of SGA births from 4.3 to 8.5% compared to 10.0% with the EPOPé curves. The adjustment for maternal and fetal characteristics avoids the over-representation of girls among SGA births, and reclassifies 4% of births. Among births reclassified as SGA, the frequency of medical and obstetrical risk factors for growth restriction, smoking (≥10 cigarettes/day), and neonatal transfer is higher than among non-SGA births (P<0.01). The EPOPé curves are more suitable for French births than currently used curves, and their adjustment improves the identification of mothers and babies at risk of growth restriction and poor perinatal outcomes. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Fitting the curve in Excel®: Systematic curve fitting of laboratory and remotely sensed planetary spectra

NASA Astrophysics Data System (ADS)

McCraig, Michael A.; Osinski, Gordon R.; Cloutis, Edward A.; Flemming, Roberta L.; Izawa, Matthew R. M.; Reddy, Vishnu; Fieber-Beyer, Sherry K.; Pompilio, Loredana; van der Meer, Freek; Berger, Jeffrey A.; Bramble, Michael S.; Applin, Daniel M.

2017-03-01

Spectroscopy in planetary science often provides the only information regarding the compositional and mineralogical make up of planetary surfaces. The methods employed when curve fitting and modelling spectra can be confusing and difficult to visualize and comprehend. Researchers who are new to working with spectra may find inadequate help or documentation in the scientific literature or in the software packages available for curve fitting. This problem also extends to the parameterization of spectra and the dissemination of derived metrics. Often, when derived metrics are reported, such as band centres, the discussion of exactly how the metrics were derived, or if there was any systematic curve fitting performed, is not included. Herein we provide both recommendations and methods for curve fitting and explanations of the terms and methods used. Techniques to curve fit spectral data of various types are demonstrated using simple-to-understand mathematics and equations written to be used in Microsoft Excel® software, free of macros, in a cut-and-paste fashion that allows one to curve fit spectra in a reasonably user-friendly manner. The procedures use empirical curve fitting, include visualizations, and ameliorates many of the unknowns one may encounter when using black-box commercial software. The provided framework is a comprehensive record of the curve fitting parameters used, the derived metrics, and is intended to be an example of a format for dissemination when curve fitting data.

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

PubMed

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

2012-09-01

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.

B-737 flight test of curved-path and steep-angle approaches using MLS guidance

NASA Technical Reports Server (NTRS)

Branstetter, J. R.; White, W. F.

1989-01-01

A series of flight tests were conducted to collect data for jet transport aircraft flying curved-path and steep-angle approaches using Microwave Landing System (MLS) guidance. During the test, 432 approaches comprising seven different curved-paths and four glidepath angles varying from 3 to 4 degrees were flown in NASA Langley's Boeing 737 aircraft (Transport Systems Research Vehicle) using an MLS ground station at the NASA Wallops Flight Facility. Subject pilots from Piedmont Airlines flew the approaches using conventional cockpit instrumentation (flight director and Horizontal Situation Indicator (HSI). The data collected will be used by FAA procedures specialists to develop standards and criteria for designing MLS terminal approach procedures (TERPS). The use of flight simulation techniques greatly aided the preliminary stages of approach development work and saved a significant amount of costly flight time. This report is intended to complement a data report to be issued by the FAA Office of Aviation Standards which will contain all detailed data analysis and statistics.

Discrete Jordan curve theorem

NASA Astrophysics Data System (ADS)

Chen, Li

1999-09-01

According to a general definition of discrete curves, surfaces, and manifolds (Li Chen, 'Generalized discrete object tracking algorithms and implementations, ' In Melter, Wu, and Latecki ed, Vision Geometry VI, SPIE Vol. 3168, pp 184 - 195, 1997.). This paper focuses on the Jordan curve theorem in 2D discrete spaces. The Jordan curve theorem says that a (simply) closed curve separates a simply connected surface into two components. Based on the definition of discrete surfaces, we give three reasonable definitions of simply connected spaces. Theoretically, these three definition shall be equivalent. We have proved the Jordan curve theorem under the third definition of simply connected spaces. The Jordan theorem shows the relationship among an object, its boundary, and its outside area. In continuous space, the boundary of an mD manifold is an (m - 1)D manifold. The similar result does apply to regular discrete manifolds. The concept of a new regular nD-cell is developed based on the regular surface point in 2D, and well-composed objects in 2D and 3D given by Latecki (L. Latecki, '3D well-composed pictures,' In Melter, Wu, and Latecki ed, Vision Geometry IV, SPIE Vol 2573, pp 196 - 203, 1995.).

Declining Rotation Curves at z = 2 in ΛCDM Galaxy Formation Simulations

NASA Astrophysics Data System (ADS)

Teklu, Adelheid F.; Remus, Rhea-Silvia; Dolag, Klaus; Arth, Alexander; Burkert, Andreas; Obreja, Aura; Schulze, Felix

2018-02-01

Selecting disk galaxies from the cosmological, hydrodynamical simulation Magneticum Pathfinder, we show that almost half of our poster child disk galaxies at z = 2 show significantly declining rotation curves and low dark matter fractions, very similar to recently reported observations. These galaxies do not show any anomalous behavior, they reside in standard dark matter halos, and they typically grow significantly in mass until z = 0, where they span all morphological classes, including disk galaxies matching present-day rotation curves and observed dark matter fractions. Our findings demonstrate that declining rotation curves and low dark matter fractions in rotation-dominated galaxies at z = 2 appear naturally within the ΛCDM paradigm and reflect the complex baryonic physics, which plays a role at the peak epoch of star formation. In addition, we find some dispersion-dominated galaxies at z = 2 that host a significant gas disk and exhibit similar shaped rotation curves as the disk galaxy population, rendering it difficult to differentiate between these two populations with currently available observation techniques.

Use of lignin extracted from different plant sources as standards in the spectrophotometric acetyl bromide lignin method.

PubMed

Fukushima, Romualdo S; Kerley, Monty S

2011-04-27

A nongravimetric acetyl bromide lignin (ABL) method was evaluated to quantify lignin concentration in a variety of plant materials. The traditional approach to lignin quantification required extraction of lignin with acidic dioxane and its isolation from each plant sample to construct a standard curve via spectrophotometric analysis. Lignin concentration was then measured in pre-extracted plant cell walls. However, this presented a methodological complexity because extraction and isolation procedures are lengthy and tedious, particularly if there are many samples involved. This work was targeted to simplify lignin quantification. Our hypothesis was that any lignin, regardless of its botanical origin, could be used to construct a standard curve for the purpose of determining lignin concentration in a variety of plants. To test our hypothesis, lignins were isolated from a range of diverse plants and, along with three commercial lignins, standard curves were built and compared among them. Slopes and intercepts derived from these standard curves were close enough to allow utilization of a mean extinction coefficient in the regression equation to estimate lignin concentration in any plant, independent of its botanical origin. Lignin quantification by use of a common regression equation obviates the steps of lignin extraction, isolation, and standard curve construction, which substantially expedites the ABL method. Acetyl bromide lignin method is a fast, convenient analytical procedure that may routinely be used to quantify lignin.

Effects of experimental design on calibration curve precision in routine analysis

PubMed Central

Pimentel, Maria Fernanda; Neto, Benício de Barros; Saldanha, Teresa Cristina B.

1998-01-01

A computational program which compares the effciencies of different experimental designs with those of maximum precision (D-optimized designs) is described. The program produces confidence interval plots for a calibration curve and provides information about the number of standard solutions, concentration levels and suitable concentration ranges to achieve an optimum calibration. Some examples of the application of this novel computational program are given, using both simulated and real data. PMID:18924816

Factors related to curved femur in elderly Japanese women

PubMed Central

Tsuchie, Hiroyuki; Miyakoshi, Naohisa; Kasukawa, Yuji; Senma, Seietsu; Narita, Yuichiro; Miyamoto, Seiya; Hatakeyama, Yuji; Sasaki, Kana; Shimada, Yoichi

2016-01-01

Background Multiple factors are involved in the development of atypical femoral fractures, and excessive curvature of the femur is thought to be one of them. However, the pathogenesis of femoral curvature is unknown. We evaluated the influence of factors related to bone metabolism and posture on the development of femoral curvature. Methods A total of 139 women participated in the present study. Curvatures were measured using antero-posterior and lateral radiography of the femur. We evaluated some bone and vitamin D metabolism markers in serum, the bone mineral density (BMD), lumbar spine alignment, and pelvic tilt. Results We divided the women into two groups, curved and non-curved groups, based on the average plus standard deviation as the cut-off between the groups. When univariate logistic regression analysis was performed to detect factors affecting femoral curvature, the following were identified as indices significantly affecting the curvature: age of the patients, serum concentrations of calcium, intact parathyroid hormone, pentosidine, homocysteine and 25-hydroxyvitamin D (25(OH)D), and BMD of the proximal femur (P < 0.05) both in the lateral and anterior curvatures. When we used multivariate analyses to assess these factors, only 25(OH)D and age (lateral and anterior standardized odds ratio: 0.776 and 0.385, and 2.312 and 4.472, respectively) affected the femoral curvature (P < 0.05). Conclusion Femoral curvature is strongly influenced by age and serum vitamin D. PMID:27228191

Analysis of variation in virulence of Beauveria bassiana against insect pests of pigeonpea using qPCR.

PubMed

Senthilraja, Govindasamy; Anand, Theerthagiri; Mohankumar, Subbarayalu; Raguchander, Thiruvengadam; Samiyappan, Ramasamy

2018-03-01

Beauveria bassiana is a broad spectrum microbial bioagent used for the control of agriculturally important insect pests. However, in our experiments, two virulent isolates of B. bassiana (B2 and B10) showed specific preference toward Maruca vitrata and Helicoverpa armigera of pigeonpea. To better understand this feature, we developed a qPCR assay to quantify the chitinase (virulence factor) of B. bassiana during the infection process. Isolates of B. bassiana were grown on insect cuticle amended medium and minimal medium (without insect cuticle) to assess the induction of chitinase. Our results revealed a positive correlation between expression of chitinase by B. bassiana and the substrates (with or without cuticles of M. vitrata and H. armigera) used. This study showcases the methodology to quantify the chitinase and analysis of variation in virulence of B. bassiana (B2 and B10) against M. vitrata and H. armigera. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Langevin Equation on Fractal Curves

NASA Astrophysics Data System (ADS)

Satin, Seema; Gangal, A. D.

2016-07-01

We analyze random motion of a particle on a fractal curve, using Langevin approach. This involves defining a new velocity in terms of mass of the fractal curve, as defined in recent work. The geometry of the fractal curve, plays an important role in this analysis. A Langevin equation with a particular model of noise is proposed and solved using techniques of the Fα-Calculus.

The Kepler Light Curve of V344 LYR: Constraining the Thermal-Viscous Limit Cycle Instability

NASA Technical Reports Server (NTRS)

Cannizzo, J. K.; Still, M. D.; Howell, S. B.; Wood, M. A.; Smale, A. P.

2010-01-01

We present time dependent modeling based on the accretion disk limit cycle model for a 90 d light curve of the short period SU UMa-type dwarf nova V344 Lyr taken by Kepler. The unprecedented precision and cadence (1 minute) far surpass that generally available for long term light curves. The data encompass a super outburst, preceded by three normal (i.e., short) outbursts and followed by two normal outbursts. The main decay of the super outburst is nearly perfectly exponential, decaying at a rate approx.12 d/mag, while the much more rapid decays of the normal outbursts exhibit a faster-than-exponential shape. We show that the standard limit cycle model can account for the light curve, without the need for either the thermal-tidal instability or enhanced mass transfer.

Nongaussian distribution curve of heterophorias among children.

PubMed

Letourneau, J E; Giroux, R

1991-02-01

The purpose of this study was to measure the distribution curve of horizontal and vertical phorias among children. Kolmogorov-Smirnov goodness of fit tests showed that these distribution curves were not Gaussian among (N = 2048) 6- to 13-year-old children. The distribution curve of horizontal phoria at far and of vertical phorias at far and at near were leptokurtic; the distribution curve of horizontal phoria at near was platykurtic. No variation of the distribution curve of heterophorias with age was observed. Comparisons of any individual findings with the general distribution curve should take the nonGaussian distribution curve of heterophorias into account.

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

PubMed

Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

2012-12-01

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rectangularization of the survival curve in The Netherlands, 1950-1992.

PubMed

Nusselder, W J; Mackenbach, J P

1996-12-01

In this article we determine whether rectangularization of the survival curve occurred in the Netherlands in the period 1950-1992. Rectangularization is defined as a trend toward a more rectangular shape of the survival curve due to increased survival and concentration of deaths around the mean age at death. We distinguish between absolute and relative rectangularization, depending on whether an increase in life expectancy is accompanied by concentration of deaths into a smaller age interval or into a smaller proportion of total life expectancy. We used measures of variability based on Keyfitz' H and the standard deviation, both life table-based. Our results show that absolute and relative rectangularization of the entire survival curve occurred in both sexes and over the complete period (except for the years 1955-1959 and 1965-1969 in men). At older ages, results differ between sexes, periods, and an absolute versus a relative definition of rectangularization. Above age 60 1/2, relative rectangularization occurred in women over the complete period and in men since 1975-1979 only, whereas absolute rectangularization occurred in both sexes since the period of 1980-1984. The implications of the recent rectangularization at older ages for achieving compression of morbidity are discussed.

Cant deficiency, curving speeds and tilt

DOT National Transportation Integrated Search

2011-01-01

In the US, increasing passenger speeds to improve trip time usually involves increasing speeds through curves. Increasing speeds through curves will increase the lateral force exerted on track during curving, thus requiring more intensive track maint...

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

Visualizing curved spacetime

NASA Astrophysics Data System (ADS)

Jonsson, Rickard M.

2005-03-01

I present a way to visualize the concept of curved spacetime. The result is a curved surface with local coordinate systems (Minkowski systems) living on it, giving the local directions of space and time. Relative to these systems, special relativity holds. The method can be used to visualize gravitational time dilation, the horizon of black holes, and cosmological models. The idea underlying the illustrations is first to specify a field of timelike four-velocities uμ. Then, at every point, one performs a coordinate transformation to a local Minkowski system comoving with the given four-velocity. In the local system, the sign of the spatial part of the metric is flipped to create a new metric of Euclidean signature. The new positive definite metric, called the absolute metric, can be covariantly related to the original Lorentzian metric. For the special case of a two-dimensional original metric, the absolute metric may be embedded in three-dimensional Euclidean space as a curved surface.

CURV 3: Characteristics and mission applications

NASA Astrophysics Data System (ADS)

Perkins, W. W.; Brady, L. K.

1984-03-01

The Cable-Controlled Underwater Recovery Vehicle (CURV) program was begun by NOSC for the specific purpose of developing economical systems to recover test ordnance at NOSC's Long Beach and San Clemente Island test ranges. CURV 3 is the latest in this series of tethered, unmanned, remotely controlled vehicles and its present capabilities far exceed the original CURV 1. Originally conceived for use as a search and recovery vehicle, CURV has evolved into a versatile and easily adaptable multipurpose work vehicle capable of performing search and recovery tasks as well as pursuing test, evaluation, exploration, and work projects. Basically, CURV is a composite of integrated subsystems including such items as propulsion, search and navigation, optics, hydraulics, and tools. Because it is unmanned and does not require life support or other complex support systems, CURV is able to perform most undersea tasks more economically and efficiently than maned systems. Also, since it is powered and controlled from the surface, CURV has a continuous, unlimited operating capability. Under emergency conditions, the vehicle can operate to 10,000-foot depths. CURV can be easily transported to any spot in the world. Upon arrival of the vehicle, control van, cable, and support gear can be mounted on a suitable ship of opportunity.

A curved edge diffraction-utilized displacement sensor for spindle metrology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, ChaBum, E-mail: clee@tntech.edu; Zhao, Rui; Jeon, Seongkyul

This paper presents a new dimensional metrological sensing principle for a curved surface based on curved edge diffraction. Spindle error measurement technology utilizes a cylindrical or spherical target artifact attached to the spindle with non-contact sensors, typically a capacitive sensor (CS) or an eddy current sensor, pointed at the artifact. However, these sensors are designed for flat surface measurement. Therefore, measuring a target with a curved surface causes error. This is due to electric fields behaving differently between a flat and curved surface than between two flat surfaces. In this study, a laser is positioned incident to the cylindrical surfacemore » of the spindle, and a photodetector collects the total field produced by the diffraction around the target surface. The proposed sensor was compared with a CS within a range of 500 μm. The discrepancy between the proposed sensor and CS was 0.017% of the full range. Its sensing performance showed a resolution of 14 nm and a drift of less than 10 nm for 7 min of operation. This sensor was also used to measure dynamic characteristics of the spindle system (natural frequency 181.8 Hz, damping ratio 0.042) and spindle runout (22.0 μm at 2000 rpm). The combined standard uncertainty was estimated as 85.9 nm under current experiment conditions. It is anticipated that this measurement technique allows for in situ health monitoring of a precision spindle system in an accurate, convenient, and low cost manner.« less

MICA: Multiple interval-based curve alignment

NASA Astrophysics Data System (ADS)

Mann, Martin; Kahle, Hans-Peter; Beck, Matthias; Bender, Bela Johannes; Spiecker, Heinrich; Backofen, Rolf

2018-01-01

MICA enables the automatic synchronization of discrete data curves. To this end, characteristic points of the curves' shapes are identified. These landmarks are used within a heuristic curve registration approach to align profile pairs by mapping similar characteristics onto each other. In combination with a progressive alignment scheme, this enables the computation of multiple curve alignments. Multiple curve alignments are needed to derive meaningful representative consensus data of measured time or data series. MICA was already successfully applied to generate representative profiles of tree growth data based on intra-annual wood density profiles or cell formation data. The MICA package provides a command-line and graphical user interface. The R interface enables the direct embedding of multiple curve alignment computation into larger analyses pipelines. Source code, binaries and documentation are freely available at https://github.com/BackofenLab/MICA

Decoding DNA labels by melting curve analysis using real-time PCR.

PubMed

Balog, József A; Fehér, Liliána Z; Puskás, László G

2017-12-01

Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

Vertebral coplanar alignment: a standardized technique for three dimensional correction in scoliosis surgery: technical description and preliminary results in Lenke type 1 curves.

PubMed

Vallespir, Gabriel Pizà; Flores, Jesús Burgos; Trigueros, Ignacio Sanpera; Sierra, Eduardo Hevia; Fernández, Pedro Doménech; Olaverri, Juan Carlos Rodríguez; Alonso, Manuel García; Galea, Rafael Ramos; Francisco, Antonio Pérez; Rodríguez de Paz, Beatriz; Carbonell, Pedro Gutiérrez; Thomas, Javier Vicente; López, José Luís González; Paulino, José Ignacio Maruenda; Pitarque, Carlos Barrios; García, Oscar Riquelme

2008-06-15

Prospective multicentric study. To present the preliminary results of an innovative method for standardized correction of scoliosis, vertebral coplanar alignment (VCA), based on a novel concept: the relocation of vertebral axis in a single plane. Normal standing spine has no rotation in coronal or transverse planes, therefore X and Z axis of vertebrae are in the same plane: they are coplanar. VCA intends to relocate these axis in one plane, correcting rotation and translation, while X axis are returned to its normal posterior divergence in sagittal plane in thoracic spine. Twenty-five consecutive adolescent idiopathic scoliosis patients (Lenke type 1) underwent posterior surgery with segmental pedicle screw fixation. Slotted tubes were attached to convex side screws. Two longitudinal rods were inserted through the end of tubes. Then, they were separated along the slots, driving the tubes into one plane, making the axis of the vertebrae coplanar and thus correcting transverse rotation and coronal translation. To obtain kyphosis, distal ends of the tubes were spread in thoracic spine. Correction was maintained by locking a definitive rod in the concave side, then tubes were retrieved and the convex side rod, inserted and tightened. Correction was assessed on preoperative and postoperative full-spine standing radiograph. Vertebral rotation was measured on computed tomography-scan and magnetic resonance imaging. Preoperative average thoracic curves of 61 degrees were corrected to 16 degrees (73%). Preoperative average thoracolumbar curves of 39 degrees were corrected to 12 degrees (70%). Preoperative average thoracic apical rotation of 24 degrees was corrected to 11 degrees (56%). Preoperative average thoracic kyphosis of 18 degrees remained unchanged after surgery; however, no patients had kyphosis <10 degrees after surgery. Rib hump improved from 30 to 11 mm (65%). There were no perioperative complications. VCA provided excellent correction of coronal and transverse

The sales learning curve.

PubMed

Leslie, Mark; Holloway, Charles A

2006-01-01

When a company launches a new product into a new market, the temptation is to immediately ramp up sales force capacity to gain customers as quickly as possible. But hiring a full sales force too early just causes the firm to burn through cash and fail to meet revenue expectations. Before it can sell an innovative product efficiently, the entire organization needs to learn how customers will acquire and use it, a process the authors call the sales learning curve. The concept of a learning curve is well understood in manufacturing. Employees transfer knowledge and experience back and forth between the production line and purchasing, manufacturing, engineering, planning, and operations. The sales learning curve unfolds similarly through the give-and-take between the company--marketing, sales, product support, and product development--and its customers. As customers adopt the product, the firm modifies both the offering and the processes associated with making and selling it. Progress along the manufacturing curve is measured by tracking cost per unit: The more a firm learns about the manufacturing process, the more efficient it becomes, and the lower the unit cost goes. Progress along the sales learning curve is measured in an analogous way: The more a company learns about the sales process, the more efficient it becomes at selling, and the higher the sales yield. As the sales yield increases, the sales learning process unfolds in three distinct phases--initiation, transition, and execution. Each phase requires a different size--and kind--of sales force and represents a different stage in a company's production, marketing, and sales strategies. Adjusting those strategies as the firm progresses along the sales learning curve allows managers to plan resource allocation more accurately, set appropriate expectations, avoid disastrous cash shortfalls, and reduce both the time and money required to turn a profit.

Anomalous D-Log E curve with high contrast developer Kodak D8 on ultra fine grain emulsion BB640.

PubMed

Ulibarrena, M; Mendez, M; Blaya, S; Fimia, A

2001-12-03

D-Log E curves, also known as H-D curves, are used since the XIX century as a tool for describing the characteristics of silver halide emulsions. This curve has a very standard shape, with a linear region, a toe, a shoulder and a solarization region. In this work we present a distortion of the usual curve due to the action of a high contrast developer, Kodak D8, on an ultra fine grain emulsion, BB640\\cite{ov04}. The solarization effect is replaced by a linear zone where developed densities increase with increasing exposures, until all silver halide present in the emulsion is reduced by developer D8 to metallic silver. Densities higher than 11 have been obtained.

Estimating time-dependent ROC curves using data under prevalent sampling.

PubMed

Li, Shanshan

2017-04-15

Prevalent sampling is frequently a convenient and economical sampling technique for the collection of time-to-event data and thus is commonly used in studies of the natural history of a disease. However, it is biased by design because it tends to recruit individuals with longer survival times. This paper considers estimation of time-dependent receiver operating characteristic curves when data are collected under prevalent sampling. To correct the sampling bias, we develop both nonparametric and semiparametric estimators using extended risk sets and the inverse probability weighting techniques. The proposed estimators are consistent and converge to Gaussian processes, while substantial bias may arise if standard estimators for right-censored data are used. To illustrate our method, we analyze data from an ovarian cancer study and estimate receiver operating characteristic curves that assess the accuracy of the composite markers in distinguishing subjects who died within 3-5 years from subjects who remained alive. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pore Size Distributions Inferred from Modified Inversion Percolation Modeling of Drainage Curves

NASA Astrophysics Data System (ADS)

Dralus, D. E.; Wang, H. F.; Strand, T. E.; Glass, R. J.; Detwiler, R. L.

2005-12-01

Experiments have been conducted of drainage in sand packs. At equilibrium, the interface between the fluids forms a saturation transition fringe where the saturation decreases monotonically with height. This behavior was observed in a 1-inch thick pack of 20-30 sand contained front and back within two thin, 12-inch-by-24-inch glass plates. The translucent chamber was illuminated from behind by a bank of fluorescent bulbs. Acquired data were in the form of images captured by a CCD camera with resolution on the grain scale. The measured intensity of the transmitted light was used to calculate the average saturation at each point in the chamber. This study used a modified invasion percolation (MIP) model to simulate the drainage experiments to evaluate the relationship between the saturation-versus-height curve at equilibrium and the pore size distribution associated with the granular medium. The simplest interpretation of a drainage curve is in terms of a distribution of capillary tubes whose radii reproduce the the observed distribution of rise heights. However, this apparent radius distribution obtained from direct inversion of the saturation profile did not yield the assumed radius distribution. Further investigation demonstrated that the equilibrium height distribution is controlled primarily by the Bond number (ratio of gravity to capillary forces) with some influence from the width of the pore radius distribution. The width of the equilibrium fringe is quantified in terms of the ratio of Bond number to the standard deviation of the pore throat distribution. The normalized saturation-vs-height curves exhibit a power-law scaling behavior consistent with both Brooks-Corey and Van Genuchten type curves. Fundamental tenets of percolation theory were used to quantify the relationship between the apparent and actual radius distributions as a function of the mean coordination number and of the ratio of Bond number to standard deviation, which was supported by both MIP

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

PubMed

de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

2016-06-01

Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

Heterozygote PCR product melting curve prediction.

PubMed

Dwight, Zachary L; Palais, Robert; Kent, Jana; Wittwer, Carl T

2014-03-01

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/. © 2013 WILEY PERIODICALS, INC.

Proficiency Standards and Cut-Scores for Language Proficiency Tests.

ERIC Educational Resources Information Center

Moy, Raymond H.

1984-01-01

Discusses the problems associated with "grading on a curve," the approach often used for standard setting on language proficiency tests. Proposes four main steps presented in the setting of a non-arbitrary cut-score. These steps not only establish a proficiency standard checked by external criteria, but also check to see that the test covers the…

Understand Centrifugal Compressor stage curves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stadler, E.L.

1986-08-01

Multistage Centrifugal Compressor Performance is generally presented in the form of a composite curve showing discharge pressure and bhp plotted as a function of capacity. This composite curve represents the cumulative performance of each stage performance curve. A simple yet quite accurate means of measuring compressor total performance is to test each stage as a single-stage compressor, usually on air with atmospheric inlets. Stage curves are then generated from the test data and three important variables are plotted: head coefficient, work coefficient and adiabatic efficiency. These variables are plotted against a normalized flow coefficient, Q/N, which is inlet volume flowmore » (cfm) divided by impeller speed (rpm). The nomenclature used to define these stage variables changes from manufacturer to manufacturer; however, the parameters presented are the same. An understanding of each parameter's theoretical derivation and determination from test data will help the engineer reviewing test curves to be more cognizant of the interrelationships between these variables; specifically, how they affect overall machine pressure rise and power consumption.« less

Analytic reflected light curves for exoplanets

NASA Astrophysics Data System (ADS)

Haggard, Hal M.; Cowan, Nicolas B.

2018-07-01

The disc-integrated reflected brightness of an exoplanet changes as a function of time due to orbital and rotational motions coupled with an inhomogeneous albedo map. We have previously derived analytic reflected light curves for spherical harmonic albedo maps in the special case of a synchronously rotating planet on an edge-on orbit (Cowan, Fuentes & Haggard). In this paper, we present analytic reflected light curves for the general case of a planet on an inclined orbit, with arbitrary spin period and non-zero obliquity. We do so for two different albedo basis maps: bright points (δ-maps), and spherical harmonics (Y_ l^m-maps). In particular, we use Wigner D-matrices to express an harmonic light curve for an arbitrary viewing geometry as a non-linear combination of harmonic light curves for the simpler edge-on, synchronously rotating geometry. These solutions will enable future exploration of the degeneracies and information content of reflected light curves, as well as fast calculation of light curves for mapping exoplanets based on time-resolved photometry. To these ends, we make available Exoplanet Analytic Reflected Lightcurves, a simple open-source code that allows rapid computation of reflected light curves.

BMI curves for preterm infants.

PubMed

Olsen, Irene E; Lawson, M Louise; Ferguson, A Nicole; Cantrell, Rebecca; Grabich, Shannon C; Zemel, Babette S; Clark, Reese H

2015-03-01

Preterm infants experience disproportionate growth failure postnatally and may be large weight for length despite being small weight for age by hospital discharge. The objective of this study was to create and validate intrauterine weight-for-length growth curves using the contemporary, large, racially diverse US birth parameters sample used to create the Olsen weight-, length-, and head-circumference-for-age curves. Data from 391 681 US infants (Pediatrix Medical Group) born at 22 to 42 weeks' gestational age (born in 1998-2006) included birth weight, length, and head circumference, estimated gestational age, and gender. Separate subsamples were used to create and validate curves. Established methods were used to determine the weight-for-length ratio that was most highly correlated with weight and uncorrelated with length. Final smoothed percentile curves (3rd to 97th) were created by the Lambda Mu Sigma (LMS) method. The validation sample was used to confirm results. The final sample included 254 454 singleton infants (57.2% male) who survived to discharge. BMI was the best overall weight-for-length ratio for both genders and a majority of gestational ages. Gender-specific BMI-for-age curves were created (n = 127 446) and successfully validated (n = 126 988). Mean z scores for the validation sample were ∼0 (∼1 SD). BMI was different across gender and gestational age. We provide a set of validated reference curves (gender-specific) to track changes in BMI for prematurely born infants cared for in the NICU for use with weight-, length-, and head-circumference-for-age intrauterine growth curves. Copyright © 2015 by the American Academy of Pediatrics.

PROPERTIES OF DUST GRAINS PROBED WITH EXTINCTION CURVES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nozawa, Takaya; Fukugita, Masataka

Modern data of the extinction curve from the ultraviolet to the near-infrared are revisited to study properties of dust grains in the Milky Way (MW) and the Small Magellanic Cloud (SMC). We confirm that the graphite-silicate mixture of grains yields the observed extinction curve with the simple power-law distribution of the grain size but with a cutoff at some maximal size: the parameters are tightly constrained to be q = 3.5 {+-} 0.2 for the size distribution a {sup -q} and the maximum radius a{sub max} = 0.24 {+-} 0.05 {mu}m, for both MW and SMC. The abundance of grains,more » and hence the elemental abundance, is constrained from the reddening versus hydrogen column density, E(B - V)/N{sub H}. If we take the solar elemental abundance as the standard for the MW, >56% of carbon should be in graphite dust, while it is <40% in the SMC using its available abundance estimate. This disparity and the relative abundance of C to Si explain the difference of the two curves. We find that 50%-60% of carbon may not necessarily be in graphite but in the amorphous or glassy phase. Iron may also be in the metallic phase or up to {approx}80% in magnetite rather than in silicates, so that the Mg/Fe ratio in astronomical olivine is arbitrary. With these substitutions, the parameters of the grain size remain unchanged. The mass density of dust grains relative to hydrogen is {rho}{sub dust}/{rho}{sub H}= 1 / (120{sup +10}{sub -16}) for the MW and 1 / (760{sup +70}{sub -90}) for the SMC under the elemental abundance constraints. We underline the importance of the wavelength dependence of the extinction curve in the near-infrared in constructing the dust model: if A{sub {lambda}}{proportional_to}{lambda}{sup -{gamma}} with {gamma} {approx_equal} 1.6, the power-law grain-size model fails, whereas it works if {gamma} {approx_equal} 1.8-2.0.« less

The roles of the binodal curve and the spinodal curve in expansions from the supercritical state with flashing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knuth, Eldon L.; Miller, David R.; Even, Uzi

2014-12-09

Data extracted from time-of-flight (TOF) measurements made on steady-state He free jets at Göttingen already in 1986 and for pulsed Ne free jets investigated recently at Tel Aviv have been added to an earlier plot of terminal condensed-phase mass fraction x{sub 2∞} as a function of the dimensionless scaling parameter Γ. Γ characterizes the source (fluid species, temperature, pressure and throat diameter); values of x{sub 2∞} are extracted from TOF measurements using conservation of energy in the free-jet expansion. For nozzles consisting of an orifice in a thin plate; the extracted data yield 22 data points which are correlated satisfactorilymore » by a single curve. The Ne free jets were expanded from a conical nozzle with a 20° half angle; the three extracted data points stand together but apart from the aforementioned curve, indicating that the presence of the conical wall influences significantly the expansion and hence the condensation. The 22 data points for the expansions via an orifice consist of 15 measurements with expansions from the gas-phase side of the binodal curve which crossed the binodal curve downstream from the sonic point and 7 measurements with expansions of the gas-phase product of the flashing which occurred after an expansion from the liquid-phase side of the binodal curve crossed the binodal curve upstream from the sonic point. The association of these 22 points with a single curve supports the alternating-phase model for flows with flashing upstream from the sonic point proposed earlier. In order to assess the role of the spinodal curve in such expansions, the spinodal curves for He and Ne were computed using general multi-parameter Helmholtz-free-energy equation-of-state formulations. Then, for the several sets of source-chamber conditions used in the free-jet measurements, thermodynamic states at key locations in the free-jet expansions (binodal curve, sonic point and spinodal curve) were evaluated, with the expansion presumed

The Aggregate Demand Curve: A Reply.

ERIC Educational Resources Information Center

Hansen, Richard B.; And Others

1987-01-01

Responds to claims about the instructional value of the downward-sloping aggregate demand curve in teaching principles of macroeconomics. Examines the effects of interest-rates and the role of money on demand curves. Concludes by arguing against the use of downward-sloping aggregate demand curves in textbooks. (RKM)

Variation in learning curves and competence for ERCP among advanced endoscopy trainees by using cumulative sum analysis.

PubMed

Wani, Sachin; Hall, Matthew; Wang, Andrew Y; DiMaio, Christopher J; Muthusamy, V Raman; Keswani, Rajesh N; Brauer, Brian C; Easler, Jeffrey J; Yen, Roy D; El Hajj, Ihab; Fukami, Norio; Ghassemi, Kourosh F; Gonzalez, Susana; Hosford, Lindsay; Hollander, Thomas G; Wilson, Robert; Kushnir, Vladimir M; Ahmad, Jawad; Murad, Faris; Prabhu, Anoop; Watson, Rabindra R; Strand, Daniel S; Amateau, Stuart K; Attwell, Augustin; Shah, Raj J; Early, Dayna; Edmundowicz, Steven A; Mullady, Daniel

2016-04-01

There are limited data on learning curves and competence in ERCP. By using a standardized data collection tool, we aimed to prospectively define learning curves and measure competence among advanced endoscopy trainees (AETs) by using cumulative sum (CUSUM) analysis. AETs were evaluated by attending endoscopists starting with the 26th hands-on ERCP examination and then every ERCP examination during the 12-month training period. A standardized ERCP competency assessment tool (using a 4-point scoring system) was used to grade the examination. CUSUM analysis was applied to produce learning curves for individual technical and cognitive components of ERCP performance (success defined as a score of 1, acceptable and unacceptable failures [p1] of 10% and 20%, respectively). Sensitivity analyses varying p1 and by using a less-stringent definition of success were performed. Five AETs were included with a total of 1049 graded ERCPs (mean ± SD, 209.8 ± 91.6/AET). The majority of cases were performed for a biliary indication (80%). The overall and native papilla allowed cannulation times were 3.1 ± 3.6 and 5.7 ± 4, respectively. Overall learning curves demonstrated substantial variability for individual technical and cognitive endpoints. Although nearly all AETs achieved competence in overall cannulation, none achieved competence for cannulation in cases with a native papilla. Sensitivity analyses increased the proportion of AETs who achieved competence. This study demonstrates that there is substantial variability in ERCP learning curves among AETs. A specific case volume does not ensure competence, especially for native papilla cannulation. Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

Energy efficiency standards and innovation

NASA Astrophysics Data System (ADS)

Morrison, Geoff

2015-01-01

Van Buskirk et al (2014 Environ. Res. Lett. 9 114010) demonstrate that the purchase price, lifecycle cost and price of improving efficiency (i.e. the incremental price of efficiency gain) decline at an accelerated rate following the adoption of the first energy efficiency standards for five consumer products. The authors show these trends using an experience curve framework (i.e. price/cost versus cumulative production). While the paper does not draw a causal link between standards and declining prices, they provide suggestive evidence using markets in the US and Europe. Below, I discuss the potential implications of the work.

Dynamic J sub I-R Curve Testing of HY-130 Steel.

DTIC Science & Technology

1981-10-01

Society for Testing and Materials 0C Degrees Celsius COD Crack-opening displacement CT Compact tension CVN Charpy V-notch dia Diameter in-lb/in 2 Inch...inches per second. A key curve for HY-130 plate was developed under dynamic loading conditions using subsized compact specimens and was applied to...face grooves were machined along the crack line to a total section reduction of 20% with a standard Charpy V-notch (CVN) cutter (450 included angle

LAMOST Spectrograph Response Curves: Stability and Application to Flux Calibration

NASA Astrophysics Data System (ADS)

Du, Bing; Luo, A.-Li; Kong, Xiao; Zhang, Jian-Nan; Guo, Yan-Xin; Cook, Neil James; Hou, Wen; Yang, Hai-Feng; Li, Yin-Bi; Song, Yi-Han; Chen, Jian-Jun; Zuo, Fang; Wu, Ke-Fei; Wang, Meng-Xin; Wu, Yue; Wang, You-Fen; Zhao, Yong-Heng

2016-12-01

The task of flux calibration for Large sky Area Multi-Object Spectroscopic Telescope (LAMOST) spectra is difficult due to many factors, such as the lack of standard stars, flat-fielding for large field of view, and variation of reddening between different stars, especially at low Galactic latitudes. Poor selection, bad spectral quality, or extinction uncertainty of standard stars not only might induce errors to the calculated spectral response curve (SRC) but also might lead to failures in producing final 1D spectra. In this paper, we inspected spectra with Galactic latitude | b| ≥slant 60^\\circ and reliable stellar parameters, determined through the LAMOST Stellar Parameter Pipeline (LASP), to study the stability of the spectrograph. To guarantee that the selected stars had been observed by each fiber, we selected 37,931 high-quality exposures of 29,000 stars from LAMOST DR2, and more than seven exposures for each fiber. We calculated the SRCs for each fiber for each exposure and calculated the statistics of SRCs for spectrographs with both the fiber variations and time variations. The result shows that the average response curve of each spectrograph (henceforth ASPSRC) is relatively stable, with statistical errors ≤10%. From the comparison between each ASPSRC and the SRCs for the same spectrograph obtained by the 2D pipeline, we find that the ASPSRCs are good enough to use for the calibration. The ASPSRCs have been applied to spectra that were abandoned by the LAMOST 2D pipeline due to the lack of standard stars, increasing the number of LAMOST spectra by 52,181 in DR2. Comparing those same targets with the Sloan Digital Sky Survey (SDSS), the relative flux differences between SDSS spectra and LAMOST spectra with the ASPSRC method are less than 10%, which underlines that the ASPSRC method is feasible for LAMOST flux calibration.

Standardization of domestic frying processes by an engineering approach.

PubMed

Franke, K; Strijowski, U

2011-05-01

An approach was developed to enable a better standardization of domestic frying of potato products. For this purpose, 5 domestic fryers differing in heating power and oil capacity were used. A very defined frying process using a highly standardized model product and a broad range of frying conditions was carried out in these fryers and the development of browning representing an important quality parameter was measured. Product-to-oil ratio, oil temperature, and frying time were varied. Quite different color changes were measured in the different fryers although the same frying process parameters were applied. The specific energy consumption for water evaporation (spECWE) during frying related to product amount was determined for all frying processes to define an engineering parameter for characterizing the frying process. A quasi-linear regression approach was applied to calculate this parameter from frying process settings and fryer properties. The high significance of the regression coefficients and a coefficient of determination close to unity confirmed the suitability of this approach. Based on this regression equation, curves for standard frying conditions (SFC curves) were calculated which describe the frying conditions required to obtain the same level of spECWE in the different domestic fryers. Comparison of browning results from the different fryers operated at conditions near the SFC curves confirmed the applicability of the approach. © 2011 Institute of Food Technologists®

Cochlear microphonic broad tuning curves

NASA Astrophysics Data System (ADS)

Ayat, Mohammad; Teal, Paul D.; Searchfield, Grant D.; Razali, Najwani

2015-12-01

It is known that the cochlear microphonic voltage exhibits much broader tuning than does the basilar membrane motion. The most commonly used explanation for this is that when an electrode is inserted at a particular point inside the scala media, the microphonic potentials of neighbouring hair cells have different phases, leading to cancelation at the electrodes location. In situ recording of functioning outer hair cells (OHCs) for investigating this hypothesis is exceptionally difficult. Therefore, to investigate the discrepancy between the tuning curves of the basilar membrane and those of the cochlear microphonic, and the effect of phase cancellation of adjacent hair cells on the broadness of the cochlear microphonic tuning curves, we use an electromechanical model of the cochlea to devise an experiment. We explore the effect of adjacent hair cells (i.e., longitudinal phase cancellation) on the broadness of the cochlear microphonic tuning curves in different locations. The results of the experiment indicate that active longitudinal coupling (i.e., coupling with active adjacent outer hair cells) only slightly changes the broadness of the CM tuning curves. The results also demonstrate that there is a π phase difference between the potentials produced by the hair bundle and the soma near the place associated with the characteristic frequency based on place-frequency maps (i.e., the best place). We suggest that the transversal phase cancellation (caused by the phase difference between the hair bundle and the soma) plays a far more important role than longitudinal phase cancellation in the broadness of the cochlear microphonic tuning curves. Moreover, by increasing the modelled longitudinal resistance resulting the cochlear microphonic curves exhibiting sharper tuning. The results of the simulations suggest that the passive network of the organ of Corti determines the phase difference between the hair bundle and soma, and hence determines the sharpness of the

Quality Quandaries: Predicting a Population of Curves

DOE PAGES

Fugate, Michael Lynn; Hamada, Michael Scott; Weaver, Brian Phillip

2017-12-19

We present a random effects spline regression model based on splines that provides an integrated approach for analyzing functional data, i.e., curves, when the shape of the curves is not parametrically specified. An analysis using this model is presented that makes inferences about a population of curves as well as features of the curves.

Quality Quandaries: Predicting a Population of Curves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fugate, Michael Lynn; Hamada, Michael Scott; Weaver, Brian Phillip

We present a random effects spline regression model based on splines that provides an integrated approach for analyzing functional data, i.e., curves, when the shape of the curves is not parametrically specified. An analysis using this model is presented that makes inferences about a population of curves as well as features of the curves.

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

PubMed

Ciulli, Sara; Pinheiro, Ana Cristina de Aguiar Saldana; Volpe, Enrico; Moscato, Michele; Jung, Tae Sung; Galeotti, Marco; Stellino, Sabrina; Farneti, Riccardo; Prosperi, Santino

2015-03-01

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

MASW on the standard seismic prospective scale using full spread recording

NASA Astrophysics Data System (ADS)

Białas, Sebastian; Majdański, Mariusz; Trzeciak, Maciej; Gałczyński, Edward; Maksym, Andrzej

2015-04-01

The Multichannel Analysis of Surface Waves (MASW) is one of seismic survey methods that use the dispersion curve of surface waves in order to describe the stiffness of the surface. Is is used mainly for geotechnical engineering scale with total length of spread between 5 - 450 m and spread offset between 1 - 100 m, the hummer is the seismic source on this surveys. The standard procedure of MASW survey is: data acquisition, dispersion analysis and inversion of extracting dispersion curve to obtain the closest theoretical curve. The final result includes share-wave velocity (Vs) values at different depth along the surveyed lines. The main goal of this work is to expand this engineering method to the bigger scale with the length of standard prospecting spread of 20 km using 4.5 Hz version of vertical component geophones. The standard vibroseis and explosive method are used as the seismic source. The acquisition were conducted on the full spread all the time during each single shoot. The seismic data acquisition used for this analysis were carried out on the Braniewo 2014 project in north of Poland. The results achieved during standard MASW procedure says that this method can be used on much bigger scale as well. The different methodology of this analysis requires only much stronger seismic source.

Viability qPCR, a new tool for Legionella risk management.

PubMed

Lizana, X; López, A; Benito, S; Agustí, G; Ríos, M; Piqué, N; Marqués, A M; Codony, F

2017-11-01

Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy. Copyright © 2017 Elsevier GmbH. All rights reserved.

QUENCH: A software package for the determination of quenching curves in Liquid Scintillation counting.

PubMed

Cassette, Philippe

2016-03-01

In Liquid Scintillation Counting (LSC), the scintillating source is part of the measurement system and its detection efficiency varies with the scintillator used, the vial and the volume and the chemistry of the sample. The detection efficiency is generally determined using a quenching curve, describing, for a specific radionuclide, the relationship between a quenching index given by the counter and the detection efficiency. A quenched set of LS standard sources are prepared by adding a quenching agent and the quenching index and detection efficiency are determined for each source. Then a simple formula is fitted to the experimental points to define the quenching curve function. The paper describes a software package specifically devoted to the determination of quenching curves with uncertainties. The experimental measurements are described by their quenching index and detection efficiency with uncertainties on both quantities. Random Gaussian fluctuations of these experimental measurements are sampled and a polynomial or logarithmic function is fitted on each fluctuation by χ(2) minimization. This Monte Carlo procedure is repeated many times and eventually the arithmetic mean and the experimental standard deviation of each parameter are calculated, together with the covariances between these parameters. Using these parameters, the detection efficiency, corresponding to an arbitrary quenching index within the measured range, can be calculated. The associated uncertainty is calculated with the law of propagation of variances, including the covariance terms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Site index curves for white fir in the southwestern United States developed using a guide curve method

Treesearch

Robert L. Mathiasen; William K. Olsen; Carleton B. Edminster

2006-01-01

Site index curves for white fir (Abies concolor) in Arizona, New Mexico, and southwestern Colorado were developed using height-age measurements and an estimated guide curve and 95% confidence intervals for individual predictions. The curves were developed using height-age data for 1,048 white firs from 263 study sites distributed across eight...

Multi-Band Light Curves from Two-Dimensional Simulations of Gamma-Ray Burst Afterglows

NASA Astrophysics Data System (ADS)

MacFadyen, Andrew

2010-01-01

The dynamics of gamma-ray burst outflows is inherently multi-dimensional. 1.) We present high resolution two-dimensional relativistic hydrodynamics simulations of GRBs in the afterglow phase using adaptive mesh refinement (AMR). Using standard synchrotron radiation models, we compute multi-band light curves, from the radio to X-ray, directly from the 2D hydrodynamics simulation data. We will present on-axis light curves for both constant density and wind media. We will also present off-axis light curves relevant for searches for orphan afterglows. We find that jet breaks are smoothed due to both off-axis viewing and wind media effects. 2.) Non-thermal radiation mechanisms in GRB afterglows require substantial magnetic field strengths. In turbulence driven by shear instabilities in relativistic magnetized gas, we demonstrate that magnetic field is naturally amplified to half a percent of the total energy (epsilon B = 0.005). We will show high resolution three dimensional relativistic MHD simulations of this process as well as particle in cell (PIC) simulations of mildly relativistic collisionless shocks.

Learning curves in health professions education.

PubMed

Pusic, Martin V; Boutis, Kathy; Hatala, Rose; Cook, David A

2015-08-01

Learning curves, which graphically show the relationship between learning effort and achievement, are common in published education research but are not often used in day-to-day educational activities. The purpose of this article is to describe the generation and analysis of learning curves and their applicability to health professions education. The authors argue that the time is right for a closer look at using learning curves-given their desirable properties-to inform both self-directed instruction by individuals and education management by instructors.A typical learning curve is made up of a measure of learning (y-axis), a measure of effort (x-axis), and a mathematical linking function. At the individual level, learning curves make manifest a single person's progress towards competence including his/her rate of learning, the inflection point where learning becomes more effortful, and the remaining distance to mastery attainment. At the group level, overlaid learning curves show the full variation of a group of learners' paths through a given learning domain. Specifically, they make overt the difference between time-based and competency-based approaches to instruction. Additionally, instructors can use learning curve information to more accurately target educational resources to those who most require them.The learning curve approach requires a fine-grained collection of data that will not be possible in all educational settings; however, the increased use of an assessment paradigm that explicitly includes effort and its link to individual achievement could result in increased learner engagement and more effective instructional design.

Dissociative Recombination without a Curve Crossing

NASA Technical Reports Server (NTRS)

Guberman, Steven L.

1994-01-01

Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

Is It Time to Change Our Reference Curve for Femur Length? Using the Z-Score to Select the Best Chart in a Chinese Population

PubMed Central

Yang, Huixia; Wei, Yumei; Su, Rina; Wang, Chen; Meng, Wenying; Wang, Yongqing; Shang, Lixin; Cai, Zhenyu; Ji, Liping; Wang, Yunfeng; Sun, Ying; Liu, Jiaxiu; Wei, Li; Sun, Yufeng; Zhang, Xueying; Luo, Tianxia; Chen, Haixia; Yu, Lijun

2016-01-01

Objective To use Z-scores to compare different charts of femur length (FL) applied to our population with the aim of identifying the most appropriate chart. Methods A retrospective study was conducted in Beijing. Fifteen hospitals in Beijing were chosen as clusters using a systemic cluster sampling method, in which 15,194 pregnant women delivered from June 20th to November 30th, 2013. The measurements of FL in the second and third trimester were recorded, as well as the last measurement obtained before delivery. Based on the inclusion and exclusion criteria, we identified FL measurements from 19996 ultrasounds from 7194 patients between 11 and 42 weeks gestation. The FL data were then transformed into Z-scores that were calculated using three series of reference equations obtained from three reports: Leung TN, Pang MW et al (2008); Chitty LS, Altman DG et al (1994); and Papageorghiou AT et al (2014). Each Z-score distribution was presented as the mean and standard deviation (SD). Skewness and kurtosis and were compared with the standard normal distribution using the Kolmogorov-Smirnov test. The histogram of their distributions was superimposed on the non-skewed standard normal curve (mean = 0, SD = 1) to provide a direct visual impression. Finally, the sensitivity and specificity of each reference chart for identifying fetuses <5th or >95th percentile (based on the observed distribution of Z-scores) were calculated. The Youden index was also listed. A scatter diagram with the 5th, 50th, and 95th percentile curves calculated from and superimposed on each reference chart was presented to provide a visual impression. Results The three Z-score distribution curves appeared to be normal, but none of them matched the expected standard normal distribution. In our study, the Papageorghiou reference curve provided the best results, with a sensitivity of 100% for identifying fetuses with measurements < 5th and > 95th percentile, and specificities of 99.9% and 81

The effect of background galaxy contamination on the absolute magnitude and light curve speed class of type Ia supernovae

NASA Technical Reports Server (NTRS)

Boisseau, John R.; Wheeler, J. Craig

1991-01-01

Observational data are presented in support of the hypothesis that background galaxy contamination is present in the photometric data of Ia supernovae and that this effect can account for the observed dispersion in the light curve speeds of most of Ia supernovae. The implication is that the observed dispersion in beta is artificial and that most of Ia supernovae have nearly homogeneous light curves. The result supports the notion that Ia supernovae are good standard candles.

Development of a New Growth Standard for Breastfed Chinese Infants: What Is the Difference from the WHO Growth Standards?

PubMed

Huang, Xiaona; Chang, Jenjen; Feng, Weiwei; Xu, Yiqun; Xu, Tao; Tang, He; Wang, Huishan; Pan, Xiaoping

2016-01-01

The objectives of this longitudinal study were to examine the trajectory of breastfed infants' growth in China to update growth standards for early childhood, and to compare these updated Chinese growth standards with the growth standards recommended by the World Health Organization (WHO) in 2006.This longitudinal cohort study enrolled 1,840 healthy breastfed infants living in an "optimal" environment favorable to growth and followed up until one year of age from 2007 to 2010. The study subjects were recruited from 60 communities in twelve cities in China. A participating infant's birth weight was measured within the first hour of the infant's life, and birth length and head circumference within 24 hours after birth. Repeated weekly and monthly anthropometric measurements were also taken. Multilevel (ML) modelling via MLwiN2.25 was fitted to estimate the growth curves of weight-for-age (WFA), length-for-age (LFA), and head circumference-for-age (HFA) for the study sample as a whole and by child sex, controlling for mode of delivery, the gravidity and parity of the mother, infant's physical measurements at birth, infant's daily food intaking frequency per day, infant's medical conditions, the season when the infant's physical measurement was taken, parents' ages, heights, and attained education, and family structure and income per month. During the first four weeks after birth, breastfed infants showed an increase in weight, length, and head circumference of 1110g, 4.9 cm, and 3.2 cm, respectively, among boys, and 980 g, 4.4 cm, and 2.8 cm, respectively, among girls. Throughout infancy, the total growth for these three was 6930 g, 26.4 cm, and 12.5 cm, respectively, among boys, and 6480 g, 25.5 cm, and 11.7 cm, respectively, among girls. As expected, there was a significant sex difference in growth during the first year. In comparison with the WHO growth standards, breastfed children in our study were heavier in weight, longer in length, and bigger in head

More Unusual Light Curves from Kepler

NASA Astrophysics Data System (ADS)

Kohler, Susanna

2017-03-01

Twenty-three new objects have been added to the growing collection of stars observed to have unusual dips in their light curves. A recent study examines these stars and the potential causes of their strange behavior.An Influx of DataThe primary Kepler mission provided light curves for over 100,000 stars, and its continuation K2 is observing another 20,000 stars every three months. As we enter an era where these enormous photometric data sets become commonplace Gaia will obtain photometry for millions of stars, and LSST billions its crucial that we understand the different categories of variability observed in these stars.The authors find three different types of light curves among their 23 unusual stars. Scallop-shell curves (top) show many undulations; persistent flux-dip class curves (middle) have discrete triangularly shaped flux dips; transient, narrow dip class curves (bottom) have only one dip that is variable in depth. The authors speculate a common cause for the scallop-shell and persistent flux-dip stars, and a different cause for the transient flux-dip stars. [Stauffer et al. 2017]After filtering out the stars with planets, those in binary systems, those with circumstellar disks, and those with starspots, a number of oddities remain: a menagerie of stars with periodic variability that cant be accounted for in these categories. Some of these stars are now famous (for instance, Boyajians star); some are lesser known. But by continuing to build up this sample of stars with unusual light curves, we have a better chance of understanding the sources of variability.Building the MenagerieTo this end, a team of scientists led by John Stauffer (Spitzer Science Center at Caltech) has recently hunted for more additions to this sample in the K2 data set. In particular, they searched through the light curves from stars in the Oph and Upper Scorpius star-forming region a data set that makes up the largest collection of high-quality light curves for low-mass, pre

Interlaboratory comparison of real-time pcr protocols for quantification of general fecal indicator bacteria

USGS Publications Warehouse

Shanks, O.C.; Sivaganesan, M.; Peed, L.; Kelty, C.A.; Blackwood, A.D.; Greene, M.R.; Noble, R.T.; Bushon, R.N.; Stelzer, E.A.; Kinzelman, J.; Anan'Eva, T.; Sinigalliano, C.; Wanless, D.; Griffith, J.; Cao, Y.; Weisberg, S.; Harwood, V.J.; Staley, C.; Oshima, K.H.; Varma, M.; Haugland, R.A.

2012-01-01

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol. ?? 2011 American Chemical Society.

Growth curve for Propionibacterium acnes.

PubMed

Hall, G S; Pratt-Rippin, K; Meisler, D M; Washington, J A; Roussel, T J; Miller, D

1994-06-01

We established growth curves for Propionibacterium acnes isolates recovered from eyes with chronic postoperative endophthalmitis. The growth curve plotted the average of the duplicate bacterial concentration against time. The generation time for P. acnes calculated from the growth curves was approximately 5.1 hours. The growth of P. acnes is slower than other anaerobic bacteria. This may account for its delayed appearance in culture of ocular specimens. It may also explain treatment failure if the concentration of an antibiotic injected into the vitreous does not remain at an effective level during the critical replicative phase of the organism.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

PubMed

Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

2017-08-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Curved and conformal high-pressure vessel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croteau, Paul F.; Kuczek, Andrzej E.; Zhao, Wenping

A high-pressure vessel is provided. The high-pressure vessel may comprise a first chamber defined at least partially by a first wall, and a second chamber defined at least partially by the first wall. The first chamber and the second chamber may form a curved contour of the high-pressure vessel. A modular tank assembly is also provided, and may comprise a first mid tube having a convex geometry. The first mid tube may be defined by a first inner wall, a curved wall extending from the first inner wall, and a second inner wall extending from the curved wall. The firstmore » inner wall may be disposed at an angle relative to the second inner wall. The first mid tube may further be defined by a short curved wall opposite the curved wall and extending from the second inner wall to the first inner wall.« less

Using Curved Crystals to Study Terrace-Width Distributions.

NASA Astrophysics Data System (ADS)

Einstein, Theodore L.

Recent experiments on curved crystals of noble and late transition metals (Ortega and Juurlink groups) have renewed interest in terrace width distributions (TWD) for vicinal surfaces. Thus, it is timely to discuss refinements of TWD analysis that are absent from the standard reviews. Rather than by Gaussians, TWDs are better described by the generalized Wigner surmise, with a power-law rise and a Gaussian decay, thereby including effects evident for weak step repulsion: skewness and peak shifts down from the mean spacing. Curved crystals allow analysis of several mean spacings with the same substrate, so that one can check the scaling with the mean width. This is important since such scaling confirms well-established theory. Failure to scale also can provide significant insights. Complicating factors can include step touching (local double-height steps), oscillatory step interactions mediated by metallic (but not topological) surface states, short-range corrections to the inverse-square step repulsion, and accounting for the offset between adjacent layers of almost all surfaces. We discuss how to deal with these issues. For in-plane misoriented steps there are formulas to describe the stiffness but not yet the strength of the elastic interstep repulsion. Supported in part by NSF-CHE 13-05892.

Robotic thyroidectomy learning curve for beginning surgeons with little or no experience of endoscopic surgery.

PubMed

Park, Jae Hyun; Lee, Jandee; Hakim, Nor Azham; Kim, Ha Yan; Kang, Sang-Wook; Jeong, Jong Ju; Nam, Kee-Hyun; Bae, Keum-Seok; Kang, Seong Joon; Chung, Woong Youn

2015-12-01

This study assessed the results of robotic thyroidectomy by fellowship-trained surgeons in their initial independent practice, and whether standard fellowship training for robotic surgery shortens the learning curve. This prospective cohort study evaluated outcomes in 125 patients who underwent robotic thyroidectomy using gasless transaxillary single-incision technique by 2 recently graduated fellowship-trained surgeons. Learning curves were analyzed by operation time, with proficiency defined as the point at which the slope of the time curve became less steep. Of the 125 patients, 113 underwent robotic less-than-total thyroidectomy, 9 underwent robotic total thyroidectomy and 3 underwent robotic total thyroidectomy with modified radical neck dissection. Mean total times for these 3 operations were 100.8 ± 20.6 minutes, 134.2 ± 38.7 minutes, and 284.7 ± 60.4 minutes, respectively. For both surgeons, the operation times gradually decreased, reaching a plateau after 20 robotic less-than-total thyroidectomies. The surgical learning curve for robotic thyroidectomy performed by recently graduated fellowship-trained surgeons with little or no experience in endoscopic surgery showed excellent results compared with those in a large series of more experienced surgeons. © 2014 Wiley Periodicals, Inc.

Time-dependent classification accuracy curve under marker-dependent sampling.

PubMed

Zhu, Zhaoyin; Wang, Xiaofei; Saha-Chaudhuri, Paramita; Kosinski, Andrzej S; George, Stephen L

2016-07-01

Evaluating the classification accuracy of a candidate biomarker signaling the onset of disease or disease status is essential for medical decision making. A good biomarker would accurately identify the patients who are likely to progress or die at a particular time in the future or who are in urgent need for active treatments. To assess the performance of a candidate biomarker, the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) are commonly used. In many cases, the standard simple random sampling (SRS) design used for biomarker validation studies is costly and inefficient. In order to improve the efficiency and reduce the cost of biomarker validation, marker-dependent sampling (MDS) may be used. In a MDS design, the selection of patients to assess true survival time is dependent on the result of a biomarker assay. In this article, we introduce a nonparametric estimator for time-dependent AUC under a MDS design. The consistency and the asymptotic normality of the proposed estimator is established. Simulation shows the unbiasedness of the proposed estimator and a significant efficiency gain of the MDS design over the SRS design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integrative neural networks model for prediction of sediment rating curve parameters for ungauged basins

NASA Astrophysics Data System (ADS)

Atieh, M.; Mehltretter, S. L.; Gharabaghi, B.; Rudra, R.

2015-12-01

One of the most uncertain modeling tasks in hydrology is the prediction of ungauged stream sediment load and concentration statistics. This study presents integrated artificial neural networks (ANN) models for prediction of sediment rating curve parameters (rating curve coefficient α and rating curve exponent β) for ungauged basins. The ANN models integrate a comprehensive list of input parameters to improve the accuracy achieved; the input parameters used include: soil, land use, topographic, climatic, and hydrometric data sets. The ANN models were trained on the randomly selected 2/3 of the dataset of 94 gauged streams in Ontario, Canada and validated on the remaining 1/3. The developed models have high correlation coefficients of 0.92 and 0.86 for α and β, respectively. The ANN model for the rating coefficient α is directly proportional to rainfall erosivity factor, soil erodibility factor, and apportionment entropy disorder index, whereas it is inversely proportional to vegetation cover and mean annual snowfall. The ANN model for the rating exponent β is directly proportional to mean annual precipitation, the apportionment entropy disorder index, main channel slope, standard deviation of daily discharge, and inversely proportional to the fraction of basin area covered by wetlands and swamps. Sediment rating curves are essential tools for the calculation of sediment load, concentration-duration curve (CDC), and concentration-duration-frequency (CDF) analysis for more accurate assessment of water quality for ungauged basins.

Customized versus population-based growth curves: prediction of low body fat percent at term corrected gestational age following preterm birth.

PubMed

Law, Tameeka L; Katikaneni, Lakshmi D; Taylor, Sarah N; Korte, Jeffrey E; Ebeling, Myla D; Wagner, Carol L; Newman, Roger B

2012-07-01

Compare customized versus population-based growth curves for identification of small-for-gestational-age (SGA) and body fat percent (BF%) among preterm infants. Prospective cohort study of 204 preterm infants classified as SGA or appropriate-for-gestational-age (AGA) by population-based and customized growth curves. BF% was determined by air-displacement plethysmography. Differences between groups were compared using bivariable and multivariable linear and logistic regression analyses. Customized curves reclassified 30% of the preterm infants as SGA. SGA infants identified by customized method only had significantly lower BF% (13.8 ± 6.0) than the AGA (16.2 ± 6.3, p = 0.02) infants and similar to the SGA infants classified by both methods (14.6 ± 6.7, p = 0.51). Customized growth curves were a significant predictor of BF% (p = 0.02), whereas population-based growth curves were not a significant independent predictor of BF% (p = 0.50) at term corrected gestational age. Customized growth potential improves the differentiation of SGA infants and low BF% compared with a standard population-based growth curve among a cohort of preterm infants.

Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance.

PubMed

Gu, Huidong; Liu, Guowen; Wang, Jian; Aubry, Anne-Françoise; Arnold, Mark E

2014-09-16

A simple procedure for selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays is reported. The correct weighting factor is determined by the relationship between the standard deviation of instrument responses (σ) and the concentrations (x). The weighting factor of 1, 1/x, or 1/x(2) should be selected if, over the entire concentration range, σ is a constant, σ(2) is proportional to x, or σ is proportional to x, respectively. For the first time, we demonstrated with detailed scientific reasoning, solid historical data, and convincing justification that 1/x(2) should always be used as the weighting factor for all bioanalytical LC-MS/MS assays. The impacts of using incorrect weighting factors on curve stability, data quality, and assay performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly. Finally, the difference between the weighting factors of 1/x(2) and 1/y(2) was discussed. All of the findings can be generalized and applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm.

Monitoring pulmonary function with superimposed pulmonary gas exchange curves from standard analyzers.

PubMed

Zar, Harvey A; Noe, Frances E; Szalados, James E; Goodrich, Michael D; Busby, Michael G

2002-01-01

A repetitive graphic display of the single breath pulmonary function can indicate changes in cardiac and pulmonary physiology brought on by clinical events. Parallel advances in computer technology and monitoring make real-time, single breath pulmonary function clinically practicable. We describe a system built from a commercially available airway gas monitor and off the shelf computer and data-acquisition hardware. Analog data for gas flow rate, O2, and CO2 concentrations are introduced into a computer through an analog-to-digital conversion board. Oxygen uptake (VO2) and carbon dioxide output (VCO2) are calculated for each breath. Inspired minus expired concentrations for O2 and CO2 are displayed simultaneously with the expired gas flow rate curve for each breath. Dead-space and alveolar ventilation are calculated for each breath and readily appreciated from the display. Graphs illustrating the function of the system are presented for the following clinical scenarios; upper airway obstruction, bronchospasm, bronchopleural fistula, pulmonary perfusion changes and inadequate oxygen delivery. This paper describes a real-time, single breath pulmonary monitoring system that displays three parameters graphed against time: expired flow rate, oxygen uptake and carbon dioxide production. This system allows for early and rapid recognition of treatable conditions that may lead to adverse events without any additional patient measurements or invasive procedures. Monitoring systems similar to the one described in this paper may lead to a higher level of patient safety without any additional patient risk.

Automated Blazar Light Curves Using Machine Learning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Spencer James

2017-07-27

This presentation describes a problem and methodology pertaining to automated blazar light curves. Namely, optical variability patterns for blazars require the construction of light curves and in order to generate the light curves, data must be filtered before processing to ensure quality.

Can anthropometry measure gender discrimination? An analysis using WHO standards to assess the growth of Bangladeshi children.

PubMed

Moestue, Helen

2009-08-01

To examine the potential of anthropometry as a tool to measure gender discrimination, with particular attention to the WHO growth standards. Surveillance data collected from 1990 to 1999 were analysed. Height-for-age Z-scores were calculated using three norms: the WHO standards, the 1978 National Center for Health Statistics (NCHS) reference and the 1990 British growth reference (UK90). Bangladesh. Boys and girls aged 6-59 months (n 504 358). The three sets of growth curves provided conflicting pictures of the relative growth of girls and boys by age and over time. Conclusions on sex differences in growth depended also on the method used to analyse the curves, be it according to the shape or the relative position of the sex-specific curves. The shapes of the WHO-generated curves uniquely implied that Bangladeshi girls faltered faster or caught up slower than boys throughout their pre-school years, a finding consistent with the literature. In contrast, analysis of the relative position of the curves suggested that girls had higher WHO Z-scores than boys below 24 months of age. Further research is needed to help establish whether and how the WHO international standards can measure gender discrimination in practice, which continues to be a serious problem in many parts of the world.

Comparison of Diagnostic Accuracy of Clinical Measures of Breast Cancer–Related Lymphedema: Area Under the Curve

PubMed Central

Smoot, Betty J.; Wong, Josephine F.; Dodd, Marylin J.

2013-01-01

Objective To compare diagnostic accuracy of measures of breast cancer–related lymphedema (BCRL). Design Cross-sectional design comparing clinical measures with the criterion standard of previous diagnosis of BCRL. Setting University of California San Francisco Translational Science Clinical Research Center. Participants Women older than 18 years and more than 6 months posttreatment for breast cancer (n=141; 70 with BCRL, 71 without BCRL). Interventions Not applicable. Main Outcome Measures Sensitivity, specificity, receiver operator characteristic curve, and area under the curve (AUC) were used to evaluate accuracy. Results A total of 141 women were categorized as having (n=70) or not having (n=71) BCRL based on past diagnosis by a health care provider, which was used as the reference standard. Analyses of ROC curves for the continuous outcomes yielded AUC of .68 to .88 (P<.001); of the physical measures bioimpedance spectroscopy yielded the highest accuracy with an AUC of .88 (95% confidence interval, .80–.96) for women whose dominant arm was the affected arm. The lowest accuracy was found using the 2-cm diagnostic cutoff score to identify previously diagnosed BCRL (AUC, .54–.65). Conclusions Our findings support the use of bioimpedance spectroscopy in the assessment of existing BCRL. Refining diagnostic cutoff values may improve accuracy of diagnosis and warrant further investigation. PMID:21440706

Water retention curve for hydrate-bearing sediments

NASA Astrophysics Data System (ADS)

Dai, Sheng; Santamarina, J. Carlos

2013-11-01

water retention curve plays a central role in numerical algorithms that model hydrate dissociation in sediments. The determination of the water retention curve for hydrate-bearing sediments faces experimental difficulties, and most studies assume constant water retention curves regardless of hydrate saturation. This study employs network model simulation to investigate the water retention curve for hydrate-bearing sediments. Results show that (1) hydrate in pores shifts the curve to higher capillary pressures and the air entry pressure increases as a power function of hydrate saturation; (2) the air entry pressure is lower in sediments with patchy rather than distributed hydrate, with higher pore size variation and pore connectivity or with lower specimen slenderness along the flow direction; and (3) smaller specimens render higher variance in computed water retention curves, especially at high water saturation Sw > 0.7. Results are relevant to other sediment pore processes such as bioclogging and mineral precipitation.

Principal curve detection in complicated graph images

NASA Astrophysics Data System (ADS)

Liu, Yuncai; Huang, Thomas S.

2001-09-01

Finding principal curves in an image is an important low level processing in computer vision and pattern recognition. Principal curves are those curves in an image that represent boundaries or contours of objects of interest. In general, a principal curve should be smooth with certain length constraint and allow either smooth or sharp turning. In this paper, we present a method that can efficiently detect principal curves in complicated map images. For a given feature image, obtained from edge detection of an intensity image or thinning operation of a pictorial map image, the feature image is first converted to a graph representation. In graph image domain, the operation of principal curve detection is performed to identify useful image features. The shortest path and directional deviation schemes are used in our algorithm os principal verve detection, which is proven to be very efficient working with real graph images.

Facilitating students' application of the integral and the area under the curve concepts in physics problems

NASA Astrophysics Data System (ADS)

Nguyen, Dong-Hai

This research project investigates the difficulties students encounter when solving physics problems involving the integral and the area under the curve concepts and the strategies to facilitate students learning to solve those types of problems. The research contexts of this project are calculus-based physics courses covering mechanics and electromagnetism. In phase I of the project, individual teaching/learning interviews were conducted with 20 students in mechanics and 15 students from the same cohort in electromagnetism. The students were asked to solve problems on several topics of mechanics and electromagnetism. These problems involved calculating physical quantities (e.g. velocity, acceleration, work, electric field, electric resistance, electric current) by integrating or finding the area under the curve of functions of related quantities (e.g. position, velocity, force, charge density, resistivity, current density). Verbal hints were provided when students made an error or were unable to proceed. A total number of 140 one-hour interviews were conducted in this phase, which provided insights into students' difficulties when solving the problems involving the integral and the area under the curve concepts and the hints to help students overcome those difficulties. In phase II of the project, tutorials were created to facilitate students' learning to solve physics problems involving the integral and the area under the curve concepts. Each tutorial consisted of a set of exercises and a protocol that incorporated the helpful hints to target the difficulties that students expressed in phase I of the project. Focus group learning interviews were conducted to test the effectiveness of the tutorials in comparison with standard learning materials (i.e. textbook problems and solutions). Overall results indicated that students learning with our tutorials outperformed students learning with standard materials in applying the integral and the area under the curve

Relationship between the curve of Spee and craniofacial variables: A regression analysis.

PubMed

Halimi, Abdelali; Benyahia, Hicham; Azeroual, Mohamed-Faouzi; Bahije, Loubna; Zaoui, Fatima

2018-06-01

The aim of this regression analysis was to identify the determining factors, which impact the curve of Spee during its genesis, its therapeutic reconstruction, and its stability, within a continuously evolving craniofacial morphology throughout life. We selected a total of 107 patients, according to the inclusion criteria. A morphological and functional clinical examination was performed for each patient: plaster models, tracing of the curve of Spee, crowding, Angle's classification, overjet and overbite were thus recorded. Then, we made a cephalometric analysis based on the standardized lateral cephalograms. In the sagittal dimension, we measured the values of angles ANB, SNA, SNB, SND, I/i; and the following distances: AoBo, I/NA, i/NB, SE and SL. In the vertical dimension, we measured the values of angles FMA, GoGn/SN, the occlusal plane, and the following distances: SAr, ArD, Ar/Con, Con/Gn, GoPo, HFP, HFA and IF. The statistical analysis was performed using the SPSS software with a significance level of 0.05. Our sample including 107 subjects was composed of 77 female patients (71.3%) and 30 male patients (27.8%) 7 hypodivergent patients (6.5%), 56 hyperdivergent patients (52.3%) and 44 normodivergent patients (41.1%). Patients' mean age was 19.35±5.95 years. The hypodivergent patients presented more pronounced curves of Spee compared to the normodivergent and the hyperdivergent populations; patients in skeletal Class I presented less pronounced curves of Spee compared to patients in skeletal Class II and Class III. These differences were non significant (P>0.05). The curve of Spee was positively and moderately correlated with Angle's classification, overjet, overbite, sellion-articulare distance, and breathing type (P<0.05). We found no correlation between age, gender and the other parameters included in the study with the curve of Spee (P>0.05). Seventy five percent (75%) of the hyperdivergent patients with an oral breathing presented an overbite of 3mm

Guidelines for application of learning/cost improvement curves

NASA Technical Reports Server (NTRS)

Delionback, L. M.

1975-01-01

The differences between the terms learning curve and improvement curve are noted, as well as the differences between the Wright system and the Crawford system. Learning curve computational techniques were reviewed along with a method to arrive at a composite learning curve for a system given detail curves either by the functional techniques classification or simply categorized by subsystem. Techniques are discussed for determination of the theoretical first unit (TFU) cost using several of the currently accepted methods. Sometimes TFU cost is referred to as simply number one cost. A tabular presentation of the various learning curve slope values is given. A discussion of the various trends in the application of learning/improvement curves and an outlook for the future are presented.

Section Curve Reconstruction and Mean-Camber Curve Extraction of a Point-Sampled Blade Surface

PubMed Central

Li, Wen-long; Xie, He; Li, Qi-dong; Zhou, Li-ping; Yin, Zhou-ping

2014-01-01

The blade is one of the most critical parts of an aviation engine, and a small change in the blade geometry may significantly affect the dynamics performance of the aviation engine. Rapid advancements in 3D scanning techniques have enabled the inspection of the blade shape using a dense and accurate point cloud. This paper proposes a new method to achieving two common tasks in blade inspection: section curve reconstruction and mean-camber curve extraction with the representation of a point cloud. The mathematical morphology is expanded and applied to restrain the effect of the measuring defects and generate an ordered sequence of 2D measured points in the section plane. Then, the energy and distance are minimized to iteratively smoothen the measured points, approximate the section curve and extract the mean-camber curve. In addition, a turbine blade is machined and scanned to observe the curvature variation, energy variation and approximation error, which demonstrates the availability of the proposed method. The proposed method is simple to implement and can be applied in aviation casting-blade finish inspection, large forging-blade allowance inspection and visual-guided robot grinding localization. PMID:25551467

Section curve reconstruction and mean-camber curve extraction of a point-sampled blade surface.

PubMed

Li, Wen-long; Xie, He; Li, Qi-dong; Zhou, Li-ping; Yin, Zhou-ping

2014-01-01

The blade is one of the most critical parts of an aviation engine, and a small change in the blade geometry may significantly affect the dynamics performance of the aviation engine. Rapid advancements in 3D scanning techniques have enabled the inspection of the blade shape using a dense and accurate point cloud. This paper proposes a new method to achieving two common tasks in blade inspection: section curve reconstruction and mean-camber curve extraction with the representation of a point cloud. The mathematical morphology is expanded and applied to restrain the effect of the measuring defects and generate an ordered sequence of 2D measured points in the section plane. Then, the energy and distance are minimized to iteratively smoothen the measured points, approximate the section curve and extract the mean-camber curve. In addition, a turbine blade is machined and scanned to observe the curvature variation, energy variation and approximation error, which demonstrates the availability of the proposed method. The proposed method is simple to implement and can be applied in aviation casting-blade finish inspection, large forging-blade allowance inspection and visual-guided robot grinding localization.

Statistical aspects of modeling the labor curve.

PubMed

Zhang, Jun; Troendle, James; Grantz, Katherine L; Reddy, Uma M

2015-06-01

In a recent review by Cohen and Friedman, several statistical questions on modeling labor curves were raised. This article illustrates that asking data to fit a preconceived model or letting a sufficiently flexible model fit observed data is the main difference in principles of statistical modeling between the original Friedman curve and our average labor curve. An evidence-based approach to construct a labor curve and establish normal values should allow the statistical model to fit observed data. In addition, the presence of the deceleration phase in the active phase of an average labor curve was questioned. Forcing a deceleration phase to be part of the labor curve may have artificially raised the speed of progression in the active phase with a particularly large impact on earlier labor between 4 and 6 cm. Finally, any labor curve is illustrative and may not be instructive in managing labor because of variations in individual labor pattern and large errors in measuring cervical dilation. With the tools commonly available, it may be more productive to establish a new partogram that takes the physiology of labor and contemporary obstetric population into account. Copyright © 2015 Elsevier Inc. All rights reserved.

Mentorship, learning curves, and balance.

PubMed

Cohen, Meryl S; Jacobs, Jeffrey P; Quintessenza, James A; Chai, Paul J; Lindberg, Harald L; Dickey, Jamie; Ungerleider, Ross M

2007-09-01

Professionals working in the arena of health care face a variety of challenges as their careers evolve and develop. In this review, we analyze the role of mentorship, learning curves, and balance in overcoming challenges that all such professionals are likely to encounter. These challenges can exist both in professional and personal life. As any professional involved in health care matures, complex professional skills must be mastered, and new professional skills must be acquired. These skills are both technical and judgmental. In most circumstances, these skills must be learned. In 2007, despite the continued need for obtaining new knowledge and learning new skills, the professional and public tolerance for a "learning curve" is much less than in previous decades. Mentorship is the key to success in these endeavours. The success of mentorship is two-sided, with responsibilities for both the mentor and the mentee. The benefits of this relationship must be bidirectional. It is the responsibility of both the student and the mentor to assure this bidirectional exchange of benefit. This relationship requires time, patience, dedication, and to some degree selflessness. This mentorship will ultimately be the best tool for mastering complex professional skills and maturing through various learning curves. Professional mentorship also requires that mentors identify and explicitly teach their mentees the relational skills and abilities inherent in learning the management of the triad of self, relationships with others, and professional responsibilities.Up to two decades ago, a learning curve was tolerated, and even expected, while professionals involved in healthcare developed the techniques that allowed for the treatment of previously untreatable diseases. Outcomes have now improved to the point that this type of learning curve is no longer acceptable to the public. Still, professionals must learn to perform and develop independence and confidence. The responsibility to

A simple Lissajous curves experimental setup

NASA Astrophysics Data System (ADS)

Şahin Kızılcık, Hasan; Damlı, Volkan

2018-05-01

The aim of this study is to develop an experimental setup to produce Lissajous curves. The setup was made using a smartphone, a powered speaker (computer speaker), a balloon, a laser pointer and a piece of mirror. Lissajous curves are formed as follows: a piece of mirror is attached to a balloon. The balloon is vibrated with the sound signal provided by the speaker that is connected to a smartphone. The laser beam is reflected off the mirror and the reflection is shaped as a Lissajous curve. Because of the intersection of two frequencies (frequency of the sound signal and natural vibration frequency of the balloon), these curves are formed. They can be used to measure the ratio of frequencies.

Random Matrix Theory and Elliptic Curves

DTIC Science & Technology

2014-11-24

distribution is unlimited. 1 ELLIPTIC CURVES AND THEIR L-FUNCTIONS 2 points on that curve. Counting rational points on curves is a field with a rich ...deficiency of zeros near the origin of the histograms in Figure 1. While as d becomes large this discretization becomes smaller and has less and less effect...order of 30), the regular oscillations seen at the origin become dominated by fluctuations of an arithmetic origin, influenced by zeros of the Riemann

Geomorphological origin of recession curves

NASA Astrophysics Data System (ADS)

Biswal, Basudev; Marani, Marco

2010-12-01

We identify a previously undetected link between the river network morphology and key recession curves properties through a conceptual-physical model of the drainage process of the riparian unconfined aquifer. We show that the power-law exponent, α, of -dQ/dt vs. Q curves is related to the power-law exponent of N(l) vs. G(l) curves (which we show to be connected to Hack's law), where l is the downstream distance from the channel heads, N(l) is the number of channel reaches exactly located at a distance l from their channel head, and G(l) is the total length of the network located at a distance greater or equal to l from channel heads. Using Digital Terrain Models and daily discharge observations from 67 US basins we find that geomorphologic α estimates match well the values obtained from recession curves analyses. Finally, we argue that the link between recession flows and network morphology points to an important role of low-flow discharges in shaping the channel network.

Estimating conditional proportion curves by regression residuals.

PubMed

Han, Bing; Lim, Nelson

2010-06-15

Researchers often derive a categorical outcome from an observed continuous measurement y. For example, human obesity status can be defined by the body mass index. They proceed to estimate the conditional proportion curve p(x) = P(ycurve p(x). We propose a fully nonparametric empirical estimator using the standardized regression residuals from the general location-scale model y=m(x) + v(x)e for the original continuous measurement. We prove the consistency of the proposed estimator in regular settings. The proposed estimator has several attractive features: it readily provides estimates for alternative cutoff values, and easily accommodates the discontinuity in p(x) caused by special cutoff. Numerical examples demonstrate that our method has better performance in mean-squared error than some alternative approaches. We also discuss a design-adjusted version of the proposed estimators to accommodate survey designs. The proposed methods are applied to data on obesity measurements among female Hispanic Americans. This new approach can be applied to other epidemiological and socio-economic research that needs to define population categories from underlying continuous measurements. Copyright (c) 2010 John Wiley & Sons, Ltd.

Fitting Richards' curve to data of diverse origins

USGS Publications Warehouse

Johnson, D.H.; Sargeant, A.B.; Allen, S.H.

1975-01-01

Published techniques for fitting data to nonlinear growth curves are briefly reviewed, most techniques require knowledge of the shape of the curve. A flexible growth curve developed by Richards (1959) is discussed as an alternative when the shape is unknown. The shape of this curve is governed by a specific parameter which can be estimated from the data. We describe in detail the fitting of a diverse set of longitudinal and cross-sectional data to Richards' growth curve for the purpose of determining the age of red fox (Vulpes vulpes) pups on the basis of right hind foot length. The fitted curve is found suitable for pups less than approximately 80 days old. The curve is extrapolated to pre-natal growth and shown to be appropriate only for about 10 days prior to birth.

The bolometric light curves and physical parameters of stripped-envelope supernovae

DOE PAGES

Prentice, S. J.; Mazzali, P. A.; Pian, E.; ...

2016-02-08

The optical and optical/near-infrared pseudo-bolometric light curves of 85 stripped-envelope supernovae (SNe) are constructed using a consistent method and a standard cosmology. The light curves are analysed to derive temporal characteristics and peak luminosity L p , enabling the construction of a luminosity function. Subsequently, the mass of 56 Ni synthesized in the explosion, along with the ratio of ejecta mass to ejecta kinetic energy, are found. Analysis shows that host-galaxy extinction is an important factor in accurately determining luminosity values as it is significantly greater than Galactic extinction in most cases. It is found that broad-lined SNe Ic (SNemore » Ic-BL) and gamma-ray burst SNe are the most luminous subtypes with a combined median L p , in erg s -1 , of log(L p) = 43.00 compared to 42.51 for SNe Ic, 42.50 for SNe Ib, and 42.36 for SNe IIb. It is also found that SNe Ic-BL synthesize approximately twice the amount of 56Ni compared with SNe Ic, Ib, and IIb, with median M Ni = 0.34, 0.16, 0.14, and 0.11 M ⊙ , respectively. SNe Ic-BL, and to a lesser extent SNe Ic, typically rise from L p /2 to L p more quickly than SNe Ib/IIb; consequently, their light curves are not as broad.« less

Applications of crude incidence curves.

PubMed

Korn, E L; Dorey, F J

1992-04-01

Crude incidence curves display the cumulative number of failures of interest as a function of time. With competing causes of failure, they are distinct from cause-specific incidence curves that treat secondary types of failures as censored observations. After briefly reviewing their definition and estimation, we present five applications of crude incidence curves to show their utility in a broad range of studies. In some of these applications it is helpful to model survival-time distributions with use of two different time metameters, for example, time from diagnosis and age of the patient. We describe how one can incorporate published vital statistics into the models when secondary types of failure correspond to common causes of death.

Assessment of opacimeter calibration according to International Standard Organization 10155.

PubMed

Gomes, J F

2001-01-01

This paper compares the calibration method for opacimeters issued by the International Standard Organization (ISO) 10155 with the manual reference method for determination of dust content in stack gases. ISO 10155 requires at least nine operational measurements, corresponding to three operational measurements per each dust emission range within the stack. The procedure is assessed by comparison with previous calibration methods for opacimeters using only two operational measurements from a set of measurements made at stacks from pulp mills. The results show that even if the international standard for opacimeter calibration requires that the calibration curve is to be obtained using 3 x 3 points, a calibration curve derived using 3 points could be, at times, acceptable in statistical terms, provided that the amplitude of individual measurements is low.

Learning curves in health care.

PubMed

Waldman, J Deane; Yourstone, Steven A; Smith, Howard L

2003-01-01

This article explores the uses of learning curve theory in medicine. Though effective application of learning curve theory in health care can result in higher quality and lower cost, it is seldom methodically applied in clinical practice. Fundamental changes are necessary in the corporate culture of medicine in order to capitalize maximally on the benefits of learning.

International transferability of accident modification functions for horizontal curves.

PubMed

Elvik, Rune

2013-10-01

Studies of the relationship between characteristics of horizontal curves and accident rate have been reported in several countries. The characteristic most often studied is the radius of a horizontal curve. Functions describing the relationship between the radius of horizontal curves and accident rate have been developed in Australia, Canada, Denmark, Germany, Great Britain, New Zealand, Norway, Portugal, Sweden, and the United States. Other characteristics of horizontal curves that have been studied include deflection angle, curve length, the presence of transition curves, super-elevation in curves and distance to adjacent curves. This paper assesses the international transferability of mathematical functions (accident modification functions) that have been developed to relate the radius of horizontal curves to their accident rate. The main research problem is whether these functions are similar, which enhances international transferability, or dissimilar, which reduces international transferability. Accident modification functions for horizontal curve radius developed in the countries listed above are synthesised. The sensitivity of the functions to other characteristics of curves than radius is examined. Accident modification functions developed in different countries have important similarities. The functions diverge with respect to accident rate in the sharpest curves. Copyright © 2013 Elsevier Ltd. All rights reserved.

GETPrime 2.0: gene- and transcript-specific qPCR primers for 13 species including polymorphisms

PubMed Central

David, Fabrice P.A.; Rougemont, Jacques; Deplancke, Bart

2017-01-01

GETPrime (http://bbcftools.epfl.ch/getprime) is a database with a web frontend providing gene- and transcript-specific, pre-computed qPCR primer pairs. The primers have been optimized for genome-wide specificity and for allowing the selective amplification of one or several splice variants of most known genes. To ease selection, primers have also been ranked according to defined criteria such as genome-wide specificity (with BLAST), amplicon size, and isoform coverage. Here, we report a major upgrade (2.0) of the database: eight new species (yeast, chicken, macaque, chimpanzee, rat, platypus, pufferfish, and Anolis carolinensis) now complement the five already included in the previous version (human, mouse, zebrafish, fly, and worm). Furthermore, the genomic reference has been updated to Ensembl v81 (while keeping earlier versions for backward compatibility) as a result of re-designing the back-end database and automating the import of relevant sections of the Ensembl database in species-independent fashion. This also allowed us to map known polymorphisms to the primers (on average three per primer for human), with the aim of reducing experimental error when targeting specific strains or individuals. Another consequence is that the inclusion of future Ensembl releases and other species has now become a relatively straightforward task. PMID:28053161

Estimated damage from the Cascadia Subduction Zone tsunami: A model comparisons using fragility curves

NASA Astrophysics Data System (ADS)

Wiebe, D. M.; Cox, D. T.; Chen, Y.; Weber, B. A.; Chen, Y.

2012-12-01

Building damage from a hypothetical Cascadia Subduction Zone tsunami was estimated using two methods and applied at the community scale. The first method applies proposed guidelines for a new ASCE 7 standard to calculate the flow depth, flow velocity, and momentum flux from a known runup limit and estimate of the total tsunami energy at the shoreline. This procedure is based on a potential energy budget, uses the energy grade line, and accounts for frictional losses. The second method utilized numerical model results from previous studies to determine maximum flow depth, velocity, and momentum flux throughout the inundation zone. The towns of Seaside and Canon Beach, Oregon, were selected for analysis due to the availability of existing data from previously published works. Fragility curves, based on the hydrodynamic features of the tsunami flow (inundation depth, flow velocity, and momentum flux) and proposed design standards from ASCE 7 were used to estimate the probability of damage to structures located within the inundations zone. The analysis proceeded at the parcel level, using tax-lot data to identify construction type (wood, steel, and reinforced-concrete) and age, which was used as a performance measure when applying the fragility curves and design standards. The overall probability of damage to civil buildings was integrated for comparison between the two methods, and also analyzed spatially for damage patterns, which could be controlled by local bathymetric features. The two methods were compared to assess the sensitivity of the results to the uncertainty in the input hydrodynamic conditions and fragility curves, and the potential advantages of each method discussed. On-going work includes coupling the results of building damage and vulnerability to an economic input output model. This model assesses trade between business sectors located inside and outside the induction zone, and is used to measure the impact to the regional economy. Results highlight

In-silico prediction of concentration-dependent viscosity curves for monoclonal antibody solutions

PubMed Central

Tomar, Dheeraj S.; Li, Li; Broulidakis, Matthew P.; Luksha, Nicholas G.; Burns, Christopher T.; Singh, Satish K.; Kumar, Sandeep

2017-01-01

ABSTRACT Early stage developability assessments of monoclonal antibody (mAb) candidates can help reduce risks and costs associated with their product development. Forecasting viscosity of highly concentrated mAb solutions is an important aspect of such developability assessments. Reliable predictions of concentration-dependent viscosity behaviors for mAb solutions in platform formulations can help screen or optimize drug candidates for flexible manufacturing and drug delivery options. Here, we present a computational method to predict concentration-dependent viscosity curves for mAbs solely from their sequence—structural attributes. This method was developed using experimental data on 16 different mAbs whose concentration-dependent viscosity curves were experimentally obtained under standardized conditions. Each concentration-dependent viscosity curve was fitted with a straight line, via logarithmic manipulations, and the values for intercept and slope were obtained. Intercept, which relates to antibody diffusivity, was found to be nearly constant. In contrast, slope, the rate of increase in solution viscosity with solute concentration, varied significantly across different mAbs, demonstrating the importance of intermolecular interactions toward viscosity. Next, several molecular descriptors for electrostatic and hydrophobic properties of the 16 mAbs derived using their full-length homology models were examined for potential correlations with the slope. An equation consisting of hydrophobic surface area of full-length antibody and charges on VH, VL, and hinge regions was found to be capable of predicting the concentration-dependent viscosity curves of the antibody solutions. Availability of this computational tool may facilitate material-free high-throughput screening of antibody candidates during early stages of drug discovery and development. PMID:28125318

In-silico prediction of concentration-dependent viscosity curves for monoclonal antibody solutions.

PubMed

Tomar, Dheeraj S; Li, Li; Broulidakis, Matthew P; Luksha, Nicholas G; Burns, Christopher T; Singh, Satish K; Kumar, Sandeep

2017-04-01

Early stage developability assessments of monoclonal antibody (mAb) candidates can help reduce risks and costs associated with their product development. Forecasting viscosity of highly concentrated mAb solutions is an important aspect of such developability assessments. Reliable predictions of concentration-dependent viscosity behaviors for mAb solutions in platform formulations can help screen or optimize drug candidates for flexible manufacturing and drug delivery options. Here, we present a computational method to predict concentration-dependent viscosity curves for mAbs solely from their sequence-structural attributes. This method was developed using experimental data on 16 different mAbs whose concentration-dependent viscosity curves were experimentally obtained under standardized conditions. Each concentration-dependent viscosity curve was fitted with a straight line, via logarithmic manipulations, and the values for intercept and slope were obtained. Intercept, which relates to antibody diffusivity, was found to be nearly constant. In contrast, slope, the rate of increase in solution viscosity with solute concentration, varied significantly across different mAbs, demonstrating the importance of intermolecular interactions toward viscosity. Next, several molecular descriptors for electrostatic and hydrophobic properties of the 16 mAbs derived using their full-length homology models were examined for potential correlations with the slope. An equation consisting of hydrophobic surface area of full-length antibody and charges on V H , V L , and hinge regions was found to be capable of predicting the concentration-dependent viscosity curves of the antibody solutions. Availability of this computational tool may facilitate material-free high-throughput screening of antibody candidates during early stages of drug discovery and development.

Surface family with a common involute asymptotic curve

NASA Astrophysics Data System (ADS)

Bayram, Ergi˙n; Bi˙li˙ci˙, Mustafa

2016-03-01

We construct a surface family possessing an involute of a given curve as an asymptotic curve. We express necessary and sufficient conditions for that curve with the above property. We also present natural results for such ruled surfaces. Finally, we illustrate the method with some examples, e.g. circles and helices as given curves.

Understanding and Taking Control of Surgical Learning Curves.

PubMed

Gofton, Wade T; Papp, Steven R; Gofton, Tyson; Beaulé, Paul E

2016-01-01

As surgical techniques continue to evolve, surgeons will have to integrate new skills into their practice. A learning curve is associated with the integration of any new procedure; therefore, it is important for surgeons who are incorporating a new technique into their practice to understand what the reported learning curve might mean for them and their patients. A learning curve should not be perceived as negative because it can indicate progress; however, surgeons need to understand how to optimize the learning curve to ensure progress with minimal patient risk. It is essential for surgeons who are implementing new procedures or skills to define potential learning curves, examine how a reported learning curve may relate to an individual surgeon's in-practice learning and performance, and suggest methods in which an individual surgeon can modify his or her specific learning curve in order to optimize surgical outcomes and patient safety. A defined personal learning contract may be a practical method for surgeons to proactively manage their individual learning curve and provide evidence of their efforts to safely improve surgical practice.

The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data

PubMed Central

Korenkova, Vlasta; Slyskova, Jana; Novosadova, Vendula; Pizzamiglio, Sara; Langerova, Lucie; Bjorkman, Jens; Vycital, Ondrej; Liska, Vaclav; Levy, Miroslav; Veskrna, Karel; Vodicka, Pavel; Vodickova, Ludmila; Kubista, Mikael; Verderio, Paolo

2016-01-01

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing. PMID:27383461

Bragg Curve, Biological Bragg Curve and Biological Issues in Space Radiation Protection with Shielding

NASA Technical Reports Server (NTRS)

Honglu, Wu; Cucinotta, F.A.; Durante, M.; Lin, Z.; Rusek, A.

2006-01-01

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LET gamma or X-rays, the presence of shielding does not always reduce the radiation risks for energetic charged particle exposure. Since the dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak, the Bragg curve does not necessarily represent the biological damage along the particle traversal since biological effects are influenced by the track structure of both primary and secondary particles. Therefore, the biological Bragg curve is dependent on the energy and the type of the primary particle, and may vary for different biological endpoints. To achieve a Bragg curve distribution, we exposed cells to energetic heavy ions with the beam geometry parallel to a monolayer of fibroblasts. Qualitative analyses of gamma-H2AX fluorescence, a known marker of DSBs, indicated increased clustering of DNA damage before the Bragg peak, enhanced homogenous distribution at the peak, and provided visual evidence of high linear energy transfer (LET) particle traversal of cells beyond the Bragg peak. A quantitative biological response curve generated for micronuclei (MN) induction across the Bragg curve did not reveal an increased yield of MN at the location of the Bragg peak. However, the ratio of mono-to bi-nucleated cells, which indicates inhibition in cell progression, increased at the Bragg peak location. These results, along with other biological concerns, show that space radiation protection with shielding can be a complicated issue.

Setting Performance Standards for Technical and Nontechnical Competence in General Surgery.

PubMed

Szasz, Peter; Bonrath, Esther M; Louridas, Marisa; Fecso, Andras B; Howe, Brett; Fehr, Adam; Ott, Michael; Mack, Lloyd A; Harris, Kenneth A; Grantcharov, Teodor P

2017-07-01

The objectives of this study were to (1) create a technical and nontechnical performance standard for the laparoscopic cholecystectomy, (2) assess the classification accuracy and (3) credibility of these standards, (4) determine a trainees' ability to meet both standards concurrently, and (5) delineate factors that predict standard acquisition. Scores on performance assessments are difficult to interpret in the absence of established standards. Trained raters observed General Surgery residents performing laparoscopic cholecystectomies using the Objective Structured Assessment of Technical Skill (OSATS) and the Objective Structured Assessment of Non-Technical Skills (OSANTS) instruments, while as also providing a global competent/noncompetent decision for each performance. The global decision was used to divide the trainees into 2 contrasting groups and the OSATS or OSANTS scores were graphed per group to determine the performance standard. Parametric statistics were used to determine classification accuracy and concurrent standard acquisition, receiver operator characteristic (ROC) curves were used to delineate predictive factors. Thirty-six trainees were observed 101 times. The technical standard was an OSATS of 21.04/35.00 and the nontechnical standard an OSANTS of 22.49/35.00. Applying these standards, competent/noncompetent trainees could be discriminated in 94% of technical and 95% of nontechnical performances (P < 0.001). A 21% discordance between technically and nontechnically competent trainees was identified (P < 0.001). ROC analysis demonstrated case experience and trainee level were both able to predict achieving the standards with an area under the curve (AUC) between 0.83 and 0.96 (P < 0.001). The present study presents defensible standards for technical and nontechnical performance. Such standards are imperative to implementing summative assessments into surgical training.

Beyond the morphology of the glucose curve following an oral glucose tolerance test in obese youth.

PubMed

Nolfe, Giuseppe; Spreghini, Maria Rita; Sforza, Rita Wietrzycowska; Morino, Giuseppe; Manco, Melania

2012-01-01

To describe the morphology of glucose curve during the oral glucose tolerance test (OGTT) and any association with glucose tolerance, insulin action and secretion in obese youth. Cross-sectional. OGTT data of 553 patients were analysed. Subjects were divided in groups based on the morphology (i.e. monophasic, biphasic, triphasic and upward monotonous) of glucose curve. Insulin action was estimated by the homeostasis model assessment of insulin resistance, the insulin sensitivity, the muscle insulin sensitivity and the hepatic insulin resistance indexes (HIRI), and the oral glucose insulin sensitivity (OGIS). Insulin secretion was estimated by the insulinogenic index (IGI). Disposition index, including the insulin secretion-sensitivity index-2, and areas under glucose (AUC(G)) and insulin (AUC(I)) curves were computed. In patients with normal glucose tolerance (n=522), prevalent morphology of the glucose curve was monophasic (n=285, 54%). Monophasic morphology was associated with the highest concentration of 1 h plasma glucose (P<0.0001) and AUC(G) (P<0.0001); biphasic morphology with better insulin sensitivity as estimated by OGIS (P<0.03) and lower AUC(I) (P<0.0001); triphasic morphology with the highest values of HIRI (P<0.02) and IGI (P<0.007). By combining morphologies of glucose and insulin curves or time of the glucose peak, a deeper characterisation of different phenotypes of glucose metabolism emerged. Morphologies of the glucose curve seem reflecting different metabolic phenotypes of insulin action and secretion, particularly when combined with morphologies of insulin curve or time of glucose peak. Such findings may deserve validation in cohort study, in which glucose metabolism would be estimated by using gold standard techniques.

Development of a New Growth Standard for Breastfed Chinese Infants: What Is the Difference from the WHO Growth Standards?

PubMed Central

Chang, Jenjen; Feng, Weiwei; Xu, Yiqun; Xu, Tao; Tang, He; Wang, Huishan; Pan, Xiaoping

2016-01-01

The objectives of this longitudinal study were to examine the trajectory of breastfed infants’ growth in China to update growth standards for early childhood, and to compare these updated Chinese growth standards with the growth standards recommended by the World Health Organization (WHO) in 2006.This longitudinal cohort study enrolled 1,840 healthy breastfed infants living in an "optimal" environment favorable to growth and followed up until one year of age from 2007 to 2010. The study subjects were recruited from 60 communities in twelve cities in China. A participating infant’s birth weight was measured within the first hour of the infant’s life, and birth length and head circumference within 24 hours after birth. Repeated weekly and monthly anthropometric measurements were also taken. Multilevel (ML) modelling via MLwiN2.25 was fitted to estimate the growth curves of weight-for-age (WFA), length-for-age (LFA), and head circumference-for-age (HFA) for the study sample as a whole and by child sex, controlling for mode of delivery, the gravidity and parity of the mother, infant’s physical measurements at birth, infant’s daily food intaking frequency per day, infant’s medical conditions, the season when the infant’s physical measurement was taken, parents’ ages, heights, and attained education, and family structure and income per month. During the first four weeks after birth, breastfed infants showed an increase in weight, length, and head circumference of 1110g, 4.9 cm, and 3.2 cm, respectively, among boys, and 980 g, 4.4 cm, and 2.8 cm, respectively, among girls. Throughout infancy, the total growth for these three was 6930 g, 26.4 cm, and 12.5 cm, respectively, among boys, and 6480 g, 25.5 cm, and 11.7 cm, respectively, among girls. As expected, there was a significant sex difference in growth during the first year. In comparison with the WHO growth standards, breastfed children in our study were heavier in weight, longer in length, and bigger

New Binary Systems With Asymmetric Light Curves

NASA Astrophysics Data System (ADS)

Virnina, Natalia A.

2010-12-01

We present the results of investigation of the light curves of 27 newly discovered binary systems. Among the examined curves, there were 10 curves with statistically significant asymmetry of maximums, according the 3σ criterion for the difference between the maximal brightness. Half of these 10 curves have a higher first maximum, another half the second one. Two of these 10 curves, USNO-B1.0 1629-0064825 = VSX J052807.9+725606 and USNO-B1.0 1586-0116785, show the largest difference between magnitudes in maxima. The star VSX J052807.9+725606 also shows the secondary minimum, which is shifted from the phase φ = 0.5. The shape of the curve argues that the physical processes of this star could be close to that of well known short periodic binary system V361 Lyr, which has a spot on the surface of one star of the system. Another star, USNO-B1.0 1586-0116785, probably has a cold spot, or several spots, in the photosphere of one of the components.

Interstellar extinction curve variations towards the inner Milky Way: a challenge to observational cosmology

NASA Astrophysics Data System (ADS)

Nataf, David M.; Gonzalez, Oscar A.; Casagrande, Luca; Zasowski, Gail; Wegg, Christopher; Wolf, Christian; Kunder, Andrea; Alonso-Garcia, Javier; Minniti, Dante; Rejkuba, Marina; Saito, Roberto K.; Valenti, Elena; Zoccali, Manuela; Poleski, Radosław; Pietrzyński, Grzegorz; Skowron, Jan; Soszyński, Igor; Szymański, Michał K.; Udalski, Andrzej; Ulaczyk, Krzysztof; Wyrzykowski, Łukasz

2016-03-01

We investigate interstellar extinction curve variations towards ˜4 deg2 of the inner Milky Way in VIJKs photometry from the OGLE-III (third phase of the Optical Gravitational Lensing Experiment) and VVV (VISTA Variables in the Via Lactea) surveys, with supporting evidence from diffuse interstellar bands and F435W, F625W photometry. We obtain independent measurements towards ˜2000 sightlines of AI, E(V - I), E(I - J) and E(J - Ks), with median precision and accuracy of 2 per cent. We find that the variations in the extinction ratios AI/E(V - I), E(I - J)/E(V - I) and E(J - Ks)/E(V - I) are large (exceeding 20 per cent), significant and positively correlated, as expected. However, both the mean values and the trends in these extinction ratios are drastically shifted from the predictions of Cardelli and Fitzpatrick, regardless of how RV is varied. Furthermore, we demonstrate that variations in the shape of the extinction curve have at least two degrees of freedom, and not one (e.g. RV), which we confirm with a principal component analysis. We derive a median value of = 13.44, which is ˜60 per cent higher than the `standard' value. We show that the Wesenheit magnitude WI = I - 1.61(I - J) is relatively impervious to extinction curve variations. Given that these extinction curves are linchpins of observational cosmology, and that it is generally assumed that RV variations correctly capture variations in the extinction curve, we argue that systematic errors in the distance ladder from studies of Type Ia supernovae and Cepheids may have been underestimated. Moreover, the reddening maps from the Planck experiment are shown to systematically overestimate dust extinction by ˜100 per cent and lack sensitivity to extinction curve variations.

Evaluation of the learning curve for external cephalic version using cumulative sum analysis.

PubMed

Kim, So Yun; Han, Jung Yeol; Chang, Eun Hye; Kwak, Dong Wook; Ahn, Hyun Kyung; Ryu, Hyun Mi; Kim, Moon Young

2017-07-01

We evaluated the learning curve for external cephalic version (ECV) using learning curve-cumulative sum (LC-CUSUM) analysis. This was a retrospective study involving 290 consecutive cases between October 2013 and March 2017. We evaluated the learning curve for ECV on nulli and over para 1 group using LC-CUSUM analysis on the assumption that 50% and 70% of ECV procedures succeeded by description a trend-line of quadratic function with reliable R 2 values. The overall success rate for ECV was 64.8% (188/290), while the success rate for nullipara and over para 1 groups was 56.2% (100/178) and 78.6% (88/112), respectively. 'H' value, that the actual failure rate does not differ from the acceptable failure rate, was -3.27 and -1.635 when considering ECV success rates of 50% and 70%, respectively. Consequently, in order to obtain a consistent 50% success rate, we would require 57 nullipara cases, and in order to obtain a consistent 70% success rate, we would require 130 nullipara cases. In contrast, 8 to 10 over para 1 cases would be required for an expected success rate of 50% and 70% on over para 1 group. Even a relatively inexperienced physician can experience success with multipara and after accumulating experience, they will manage nullipara cases. Further research is required for LC-CUSUM involving several practitioners instead of a single practitioner. This will lead to the gradual implementation of standard learning curve guidelines for ECV.

Apollo 16/AS-511/LM-11 operational calibration curves. Volume 1: Calibration curves for command service module CSM 113

NASA Technical Reports Server (NTRS)

Demoss, J. F. (Compiler)

1971-01-01

Calibration curves for the Apollo 16 command service module pulse code modulation downlink and onboard display are presented. Subjects discussed are: (1) measurement calibration curve format, (2) measurement identification, (3) multi-mode calibration data summary, (4) pulse code modulation bilevel events listing, and (5) calibration curves for instrumentation downlink and meter link.

A retrospective analysis of compact fluorescent lamp experience curves and their correlations to deployment programs

DOE PAGES

Smith, Sarah Josephine; Wei, Max; Sohn, Michael D.

2016-09-17

Experience curves are useful for understanding technology development and can aid in the design and analysis of market transformation programs. Here, we employ a novel approach to create experience curves, to examine both global and North American compact fluorescent lamp (CFL) data for the years 1990–2007. We move away from the prevailing method of fitting a single, constant, exponential curve to data and instead search for break points where changes in the learning rate may have occurred. Our analysis suggests a learning rate of approximately 21% for the period of 1990–1997, and 51% and 79% in global and North Americanmore » datasets, respectively, after 1998. We use price data for this analysis; therefore our learning rates encompass developments beyond typical “learning by doing”, including supply chain impacts such as market competition. We examine correlations between North American learning rates and the initiation of new programs, abrupt technological advances, and economic and political events, and find an increased learning rate associated with design advancements and federal standards programs. Our findings support the use of segmented experience curves for retrospective and prospective technology analysis, and may imply that investments in technology programs have contributed to an increase of the CFL learning rate.« less

A retrospective analysis of compact fluorescent lamp experience curves and their correlations to deployment programs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Sarah Josephine; Wei, Max; Sohn, Michael D.

Experience curves are useful for understanding technology development and can aid in the design and analysis of market transformation programs. Here, we employ a novel approach to create experience curves, to examine both global and North American compact fluorescent lamp (CFL) data for the years 1990–2007. We move away from the prevailing method of fitting a single, constant, exponential curve to data and instead search for break points where changes in the learning rate may have occurred. Our analysis suggests a learning rate of approximately 21% for the period of 1990–1997, and 51% and 79% in global and North Americanmore » datasets, respectively, after 1998. We use price data for this analysis; therefore our learning rates encompass developments beyond typical “learning by doing”, including supply chain impacts such as market competition. We examine correlations between North American learning rates and the initiation of new programs, abrupt technological advances, and economic and political events, and find an increased learning rate associated with design advancements and federal standards programs. Our findings support the use of segmented experience curves for retrospective and prospective technology analysis, and may imply that investments in technology programs have contributed to an increase of the CFL learning rate.« less