Biomolecular Detection and Quantification最新文献_第5页

Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B 同质和数字接近结扎法检测艰难梭菌毒素A和B

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.06.003

Harvinder S. Dhillon , Gemma Johnson , Mark Shannon , Christina Greenwood , Doug Roberts , Stephen Bustin

{"title":"Homogeneous and digital proximity ligation assays for the detection of Clostridium difficile toxins A and B","authors":"Harvinder S. Dhillon , Gemma Johnson , Mark Shannon , Christina Greenwood , Doug Roberts , Stephen Bustin","doi":"10.1016/j.bdq.2016.06.003","DOIUrl":"10.1016/j.bdq.2016.06.003","url":null,"abstract":"<div><h3>Background</h3><p>The proximity ligation assay (PLA) detects proteins via their interaction with pairs of proximity probes, which are antibodies coupled to noncomplementary DNA oligonucleotides. The binding of both proximity probes to their epitopes on the target protein brings the oligonucleotides together, allowing them to be bridged by a third oligonucleotide with complementarity to the other two. This enables their ligation and the detection of the resulting amplicon by real-time quantitative PCR (qPCR), which acts as a surrogate marker for the protein of interest. Hence PLA has potential as a clinically relevant diagnostic tool for the detection of pathogens where nucleic acid based tests are inconclusive proof of infection.</p></div><div><h3>Methods</h3><p>We prepared monoclonal and polyclonal proximity probes targeting <em><em>Clostridium</em> difficile</em> toxins A (TcdA) and B (TcdB) and used hydrolysis probe-based qPCR and digital PCR (dPCR) assays to detect antibody/antigen interactions.</p></div><div><h3>Results</h3><p>The performance of the PLA assays was antibody-dependent but both TcdA and TcdB assays were more sensitive than comparable ELISAs in either single- or dualplex formats. Both PLAs could be performed using single monoclonal antibodies coupled to different oligonucleotides. Finally, we used dPCR to demonstrate its potential for accurate and reliable quantification of TcdA.</p></div><div><h3>Conclusions</h3><p>PLA with either qPCR or dPCR readout have potential as new diagnostic applications for the detection of pathogens where nucleic acid based tests do not indicate viability or expression of toxins. Importantly, since it is not always necessary to use two different antibodies, the pool of potential antibodies useful for PLA diagnostic assays is usefully enhanced.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":"10 ","pages":"Pages 2-8"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2016.06.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54134162","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: 14

Digital polymerase chain reaction for characterisation of DNA reference materials 数字聚合酶链反应用于鉴定DNA参比物

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.04.001

Somanath Bhat, Kerry R. Emslie

引用次数: 27

Applicability of digital PCR to the investigation of pediatric-onset genetic disorders 数字PCR在儿科发病遗传疾病调查中的适用性

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.06.002

Matthew E.R. Butchbach

{"title":"Applicability of digital PCR to the investigation of pediatric-onset genetic disorders","authors":"Matthew E.R. Butchbach","doi":"10.1016/j.bdq.2016.06.002","DOIUrl":"10.1016/j.bdq.2016.06.002","url":null,"abstract":"<div><p>Early-onset rare diseases have a strong impact on child healthcare even though the incidence of each of these diseases is relatively low. In order to better manage the care of these children, it is imperative to quickly diagnose the molecular bases for these disorders as well as to develop technologies with prognostic potential. Digital PCR (dPCR) is well suited for this role by providing an absolute quantification of the target DNA within a sample. This review illustrates how dPCR can be used to identify genes associated with pediatric-onset disorders, to identify copy number status of important disease-causing genes and variants and to quantify modifier genes. It is also a powerful technology to track changes in genomic biomarkers with disease progression. Based on its capability to accurately and reliably detect genomic alterations with high sensitivity and a large dynamic detection range, dPCR has the potential to become the tool of choice for the verification of pediatric disease-associated mutations identified by next generation sequencing, copy number determination and noninvasive prenatal screening.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":"10 ","pages":"Pages 9-14"},"PeriodicalIF":0.0,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2016.06.002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54134136","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: 12

Digital PCR dynamic range is approaching that of real-time quantitative PCR 数字PCR的动态范围正在接近实时定量PCR

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.10.001

Gerwyn M. Jones , Eloise Busby , Jeremy A. Garson , Paul R. Grant , Eleni Nastouli , Alison S. Devonshire , Alexandra S. Whale

引用次数: 33

Three-color crystal digital PCR 三色晶体数字PCR

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.10.002

J. Madic , A. Zocevic , V. Senlis , E. Fradet , B. Andre , S. Muller , R. Dangla , M.E. Droniou

引用次数: 90

Digital PCR, a technique for the future 数字PCR，未来的技术

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.10.003

-->Valerie Taly, Jim Huggett

引用次数: 3

Designing and interpretation of digital assays: Concentration of target in the sample and in the source of sample 数字分析的设计和解释:样品和样品源中目标物的浓度

Biomolecular Detection and Quantification Pub Date : 2016-12-01 DOI: 10.1016/j.bdq.2016.04.002

Pawel R. Debski , Piotr Garstecki

引用次数: 11

Science in the UK − whereto now? 英国的科学——现在何去何从?

Biomolecular Detection and Quantification Pub Date : 2016-09-01 DOI: 10.1016/j.bdq.2016.08.001

Stephen Bustin

引用次数: 1

Flexible analysis of digital PCR experiments using generalized linear mixed models 用广义线性混合模型灵活分析数字PCR实验

Biomolecular Detection and Quantification Pub Date : 2016-09-01 DOI: 10.1016/j.bdq.2016.06.001

Matthijs Vynck , Jo Vandesompele , Nele Nijs , Björn Menten , Ariane De Ganck , Olivier Thas

引用次数: 13

Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA 引物、探针化学及扩增靶点对病毒RNA逆转录数字PCR定量的影响

Biomolecular Detection and Quantification Pub Date : 2016-09-01 DOI: 10.1016/j.bdq.2016.08.003

Fran Van Heuverswyn, Maria Karczmarczyk , Heinz Schimmel, Stefanie Trapmann, Hendrik Emons

{"title":"Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA","authors":"Fran Van Heuverswyn, Maria Karczmarczyk , Heinz Schimmel, Stefanie Trapmann, Hendrik Emons","doi":"10.1016/j.bdq.2016.08.003","DOIUrl":"10.1016/j.bdq.2016.08.003","url":null,"abstract":"<div><p>Compared to other PCR technologies, digital PCR is a potentially highly accurate approach for the quantification of nucleic acid fragments. This study describes the impact of four experimental factors, namely primer and probe chemistry, PCR amplification target, duplexing, and template type, on the measurement results obtained by reverse transcription digital PCR (RT-dPCR) of viral RNA using <em>influenza A</em> virus as a model. Along conventional dual labelled probes (DLP), alternative primer and probe chemistries, including Zip Nucleic Acids (ZNAs), Locked Nucleic Acids (LNAs), and Scorpions<sup>®</sup>, were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (<em>In vitro</em> transcribed RNA).</p><p>While apparently duplexing or a different PCR target choice did not have a significant influence on the estimated RNA copy numbers, the impact of the choice of primer and probe chemistry and template type differed significantly for some methods. The combined standard uncertainty of the dPCR analysis results has been assessed, taking into account both the repeatability and the intermediate precision of the procedure.</p><p>Our data highlight the importance of dPCR method optimisation and the advantage of using a more sophisticated primer and probe chemistry, which turned out to be dependent on the template type. Considerations are provided with respect to the molecular diagnostics of viral RNA pathogens, and more specifically, for precise quantification of RNA, which is of tremendous importance for the development of RNA calibration materials and the qualification of these calibrants as certified reference materials.</p></div>","PeriodicalId":38073,"journal":{"name":"Biomolecular Detection and Quantification","volume":"9 ","pages":"Pages 20-28"},"PeriodicalIF":0.0,"publicationDate":"2016-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.bdq.2016.08.003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"54133817","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: 2