Biomolecular Detection and Quantification最新文献_第9页

qPCR, dPCR, NGS – A journey qPCR, dPCR, NGS -一段旅程

Biomolecular Detection and Quantification Pub Date : 2015-03-01 DOI: 10.1016/j.bdq.2015.01.001

Jim F. Huggett , Justin O’Grady, Stephen Bustin

引用次数: 16

How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments PCR效率评估有多好:精确和稳健的qPCR效率评估建议

Biomolecular Detection and Quantification Pub Date : 2015-03-01 DOI: 10.1016/j.bdq.2015.01.005

David Svec , Ales Tichopad , Vendula Novosadova , Michael W. Pfaffl , Mikael Kubista

{"title":"How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments","authors":"David Svec , Ales Tichopad , Vendula Novosadova , Michael W. Pfaffl , Mikael Kubista","doi":"10.1016/j.bdq.2015.01.005","DOIUrl":"10.1016/j.bdq.2015.01.005","url":null,"abstract":"<div><p>We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1–16 qPCR replicates per concentration and we tested 2–10μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3–4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2015.01.005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"34400267","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 362

Multi-template polymerase chain reaction 多模板聚合酶链反应

Biomolecular Detection and Quantification Pub Date : 2014-12-01 DOI: 10.1016/j.bdq.2014.11.002

Elena Kalle , Mikael Kubista , Christopher Rensing

{"title":"Multi-template polymerase chain reaction","authors":"Elena Kalle , Mikael Kubista , Christopher Rensing","doi":"10.1016/j.bdq.2014.11.002","DOIUrl":"10.1016/j.bdq.2014.11.002","url":null,"abstract":"<div><p>PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2014.11.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54133924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 82

The reproducibility of biomedical research: Sleepers awake! 生物医学研究的可重复性:睡眠者醒了!

Biomolecular Detection and Quantification Pub Date : 2014-12-01 DOI: 10.1016/j.bdq.2015.01.002

Stephen A. Bustin

引用次数: 52

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges 肠沙门氏菌血清型伤寒非经典喹诺酮类药物耐药的特征:gyrB基因的新突变和诊断挑战的报告

Biomolecular Detection and Quantification Pub Date : 2014-12-01 DOI: 10.1016/j.bdq.2015.01.003

Ruchi Gupta , Rajni Gaind , John Wain , Monorama Deb , Laishram Chandreshwor Singh , Seemi Farhat Basir

{"title":"Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges","authors":"Ruchi Gupta , Rajni Gaind , John Wain , Monorama Deb , Laishram Chandreshwor Singh , Seemi Farhat Basir","doi":"10.1016/j.bdq.2015.01.003","DOIUrl":"10.1016/j.bdq.2015.01.003","url":null,"abstract":"<div><h3>Objective</h3><p>To establish the relative importance of <em>Salmonella enterica</em> serovar Typhi with non-classical quinolone resistance.</p></div><div><h3>Methods</h3><p>Eight hundred and ninety-one isolates of <em>S.</em> Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NAL<sup>S</sup> (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from <em>gyrA</em>, <em>gyrB</em>, <em>parC</em> and <em>parE</em> and from the plasmid borne determinants: <em>qnrA</em>,B,S; aac(6′)-Ib-cr and <em>qepA</em>. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci.</p></div><div><h3>Results</h3><p>Eighty isolates with a nalidixic acid MIC of <32mg/L (NAL<sup>S</sup>) and a ciprofloxacin MIC of >0.064mg/L CIP<sup>I</sup> (ciprofloxacin reduced susceptibility) were found. In 36 NAL<sup>S</sup> CIP<sup>I</sup> isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (<em>n</em>    =2) NAL MIC of 16mg/L and CIP MIC of 0.25–0.38mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA.</p></div><div><h3>Conclusion</h3><p>The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in <em>S.</em> Typhi misses CIP<sup>I</sup>-NAL<sup>S</sup> isolates, an established phenotype in India.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2015.01.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54134012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods 一种评估实时定量环介导等温扩增方法性能的新方法

Biomolecular Detection and Quantification Pub Date : 2014-12-01 DOI: 10.1016/j.bdq.2014.11.001

Gavin J. Nixon , Helle F. Svenstrup , Carol E. Donald , Caroline Carder , Judith M. Stephenson , Stephen Morris-Jones , Jim F. Huggett , Carole A. Foy

{"title":"A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods","authors":"Gavin J. Nixon , Helle F. Svenstrup , Carol E. Donald , Caroline Carder , Judith M. Stephenson , Stephen Morris-Jones , Jim F. Huggett , Carole A. Foy","doi":"10.1016/j.bdq.2014.11.001","DOIUrl":"10.1016/j.bdq.2014.11.001","url":null,"abstract":"<div><p>Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2014.11.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54133513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 28

Making standards for quantitative real-time pneumococcal PCR 制定实时肺炎球菌定量PCR标准

Biomolecular Detection and Quantification Pub Date : 2014-12-01 DOI: 10.1016/j.bdq.2014.11.003

Susan C. Morpeth , Jim F. Huggett , David R. Murdoch , J. Anthony G. Scott

引用次数: 8

A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens 商业上可用的核酸诊断方法检测和鉴定人类肠胃炎病原体的当前概述

Biomolecular Detection and Quantification Pub Date : 2014-09-01 DOI: 10.1016/j.bdq.2014.07.001

Kate Reddington, Nina Tuite, Elizabeth Minogue, Thomas Barry

{"title":"A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens","authors":"Kate Reddington, Nina Tuite, Elizabeth Minogue, Thomas Barry","doi":"10.1016/j.bdq.2014.07.001","DOIUrl":"10.1016/j.bdq.2014.07.001","url":null,"abstract":"<div><h3>Purpose of review</h3><p>Gastroenteritis is caused by a wide range of viral, bacterial and parasitic pathogens and causes millions of deaths worldwide each year, particularly in infant populations in developing countries. Traditional microbiological culture and immunological based tests are time consuming, laborious and often lack diagnostic specificity and sensitivity. As a result patients can receive suboptimal and/or inappropriate antimicrobial treatment. In recent years, rapid nucleic acid diagnostics (NAD) technologies have become available to complement or even bypass and replace these traditional microbiological culture and immunological based tests.</p><p>The main purpose of this review is to describe a number of recently available multiparametric commercial tests, to support the rapid and accurate clinical diagnosis of human gastroenteritis. These state of the art technologies have the ability to identify a wide range of microorganisms associated with enteric gastroenteritis. Following further technological innovation and more comprehensive clinical validation studies, these NAD tests have the potential to impact on the economic burden of health care systems. These rapid NAD tests can also be used to guide improved patient therapy in a timely manner which will reduce the extent of morbidity and mortality associated with these infections globally.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2014.07.001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54133367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 52

Digital PCR: A brief history 数字PCR:简史

Biomolecular Detection and Quantification Pub Date : 2014-09-01 DOI: 10.1016/j.bdq.2014.06.001

Alexander A. Morley

引用次数: 117

A survey of tools for the analysis of quantitative PCR (qPCR) data 定量PCR (qPCR)数据分析工具综述

Biomolecular Detection and Quantification Pub Date : 2014-09-01 DOI: 10.1016/j.bdq.2014.08.002

Stephan Pabinger , Stefan Rödiger , Albert Kriegner , Klemens Vierlinger , Andreas Weinhäusel

{"title":"A survey of tools for the analysis of quantitative PCR (qPCR) data","authors":"Stephan Pabinger , Stefan Rödiger , Albert Kriegner , Klemens Vierlinger , Andreas Weinhäusel","doi":"10.1016/j.bdq.2014.08.002","DOIUrl":"10.1016/j.bdq.2014.08.002","url":null,"abstract":"<div><p>Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions.</p><p>Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR.</p><p>Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2014-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2014.08.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54133427","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 157