83 results match your criteria Biomolecular detection and quantification[Journal]


Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179.

Biomol Detect Quantif 2019 Mar 11;17:100076. Epub 2019 Feb 11.

Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764 Oberschleißheim, Germany.

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446038PMC

Editorial.

Authors:
Michael W Pfaffl

Biomol Detect Quantif 2019 Mar 22;17:100086. Epub 2019 Mar 22.

Animal Physiology & Immunology, School of Life Sciences, Technical University of Munich, Weihenstephaner Berg 3, 85354, Freising, Germany.

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http://dx.doi.org/10.1016/j.bdq.2019.100086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433997PMC

The emerging role of cell-free DNA as a molecular marker for cancer management.

Biomol Detect Quantif 2019 Mar 18;17:100087. Epub 2019 Mar 18.

Institute for Laboratory Medicine, German Heart Centre, Technical University Munich, Lazarettstraße. 36, D-80636, Munich, Germany.

An increasing number of studies demonstrate the potential use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. However, harnessing the full potential of cfDNA requires (i) the optimization and standardization of preanalytical steps, (ii) refinement of current analysis strategies, and, perhaps most importantly, (iii) significant improvements in our understanding of its origin, physical properties, and dynamics in circulation. The latter knowledge is crucial for interpreting the associations between changes in the baseline characteristics of cfDNA and the clinical manifestations of cancer. Read More

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http://dx.doi.org/10.1016/j.bdq.2019.100087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425120PMC

Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method.

Biomol Detect Quantif 2019 Mar 16;17:100083. Epub 2019 Mar 16.

Physikalisch-Technische Bundesanstalt, Abbestr. 2-12, 10587 Berlin, Germany.

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Read More

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http://dx.doi.org/10.1016/j.bdq.2019.100083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423501PMC

Next-generation sequencing of HIV-1 single genome amplicons.

Biomol Detect Quantif 2019 Mar 11;17:100080. Epub 2019 Mar 11.

U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, United States.

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. Read More

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http://dx.doi.org/10.1016/j.bdq.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423504PMC
March 2019
1 Read

Considerations and quality controls when analyzing cell-free tumor DNA.

Biomol Detect Quantif 2019 Mar 13;17:100078. Epub 2019 Feb 13.

Sahlgrenska Cancer Center, Department of Pathology and Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 1F, 413 90, Gothenburg, Sweden.

Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416156PMC

Challenging the proposed causes of the PCR plateau phase.

Biomol Detect Quantif 2019 Mar 2;17:100082. Epub 2019 Mar 2.

Applied Microbiology, Department of Chemistry, Lund University, Lund, Sweden.

Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. Read More

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http://dx.doi.org/10.1016/j.bdq.2019.100082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403077PMC
March 2019
1 Read

Corrigendum to "Incidence and detection of Beak and Feather disease virus in psittacine birds in the UAE" [Biomol. Detect. Quantif. 6 (January) (2016) 27-32].

Biomol Detect Quantif 2019 Mar 1;17:100079. Epub 2019 Mar 1.

Molecular Biology and Genetics Laboratory, P.O. Box 597, Dubai, United Arab Emirates.

[This corrects the article DOI: 10.1016/j.bdq. Read More

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http://dx.doi.org/10.1016/j.bdq.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402373PMC

Shedding light: The importance of reverse transcription efficiency standards in data interpretation.

Biomol Detect Quantif 2019 Mar 12;17:100077. Epub 2019 Feb 12.

Australian Institute for Bioengineering and Nanotechnology, Building 75, Corner College and Cooper Roads, The University of Queensland, St Lucia 4067 QLD, Australia.

The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ability to do quantitative transcriptomics analyses. Multiple studies have focused on ways to optimise this process, resulting in contradictory recommendations. Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method's impact on subsequent data analysis. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374950PMC
March 2019
1 Read

Essential role of polymerases for assay performance - Impact of polymerase replacement in a well-established assay.

Biomol Detect Quantif 2018 Dec 14;16:12-20. Epub 2018 Nov 14.

Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinaerplatz 1, 1210, Vienna, Austria.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287537PMC
December 2018

Using ddPCR to assess the DNA yield of FFPE samples.

Biomol Detect Quantif 2018 Dec 7;16:5-11. Epub 2018 Nov 7.

Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Objectives: Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287546PMC
December 2018
4 Reads

Algorithms for automated detection of hook effect-bearing amplification curves.

Biomol Detect Quantif 2018 Dec 16;16:1-4. Epub 2018 Oct 16.

Institute of Biotechnology, Brandenburg University of Technology Cottbus - Senftenberg, Senftenberg, Germany.

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287529PMC
December 2018
1 Read

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

Biomol Detect Quantif 2018 May 9;15:24-29. Epub 2018 Apr 9.

Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main Street, Winnipeg, Manitoba, Canada.

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006385PMC

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples.

Biomol Detect Quantif 2018 May 16;15:18-23. Epub 2018 Mar 16.

Population Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9, Canada.

Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006387PMC
May 2018
24 Reads

Improving the standardization of mRNA measurement by RT-qPCR.

Biomol Detect Quantif 2018 May 16;15:13-17. Epub 2018 Mar 16.

School of Biosciences, Cardiff University, Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK.

Human health and safety depend on reliable measurements in medical diagnosis and on tests that support the selection and evaluation of therapeutic intervention and newly discovered molecular biomarkers must pass a rigorous evaluation process if they are to be of benefit to patients. Measurement standardization helps to maximize data quality and confidence and ultimately improves the reproducibility of published research. Failure to consider how a given experiment may be standardized can be costly, both financially as well as in time and failure to perform and report pre-clinical research in an appropriately rigorous manner will hinder the development of diagnostic methods. Read More

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http://dx.doi.org/10.1016/j.bdq.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006386PMC

An optimized targeted Next-Generation Sequencing approach for sensitive detection of single nucleotide variants.

Biomol Detect Quantif 2018 May 9;15:6-12. Epub 2018 Jan 9.

Universitätsklinikum Carl Gustav Carus, Medizinische Klinik und Poliklinik I, Dresden, Germany.

Monitoring of minimal residual disease (MRD) has become an important clinical aspect for early relapse detection during follow-up care after cancer treatment. Still, the sensitive detection of single base pair point mutations via Next-Generation Sequencing (NGS) is hampered mainly due to high substitution error rates. We evaluated the use of NGS for the detection of low-level variants on an Ion Torrent PGM system. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766748PMC
May 2018
3 Reads

Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers.

Biomol Detect Quantif 2018 May 18;15:1-5. Epub 2017 Dec 18.

Departments of Vascular Medicine, University of Amsterdam, Amsterdam, The Netherlands.

Since the discovery of microRNAs (miRNAs), circulating miRNAs have been proposed as biomarkers for disease. Consequently, many groups have tried to identify circulating miRNA biomarkers for various types of diseases including cardiovascular disease and cancer. However, the replicability of these experiments has been disappointingly low. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737945PMC
May 2018
2 Reads

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR).

Biomol Detect Quantif 2017 Dec 1;14:7-18. Epub 2017 Nov 1.

Academic Medical Center, Department of Medical Biology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands.

Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to C or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727009PMC
December 2017

Next-generation sequencing applications in clinical bacteriology.

Biomol Detect Quantif 2017 Dec 23;14:1-6. Epub 2017 Oct 23.

Department of Health System Management, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

With the rapid advances in next generation sequencing (NGS) technologies, clinical and public health microbiology laboratories are increasingly adopting NGS technology in their workflows into their existing diagnostic cycles. In this bacteriology focused review, we review aspects and considerations for applying NGS in the clinical microbiology settings, and highlight the impact of such implementation on the analytical and post-analytical stages of diagnosis. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727008PMC
December 2017
1 Read

qPCR primer design revisited.

Biomol Detect Quantif 2017 Dec 22;14:19-28. Epub 2017 Nov 22.

Molecular and Cell Biology Team, LGC, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.

Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702850PMC
December 2017
2 Reads

Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR.

Biomol Detect Quantif 2017 Sep 20;13:40-48. Epub 2017 Sep 20.

Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium.

Introduction: For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation c. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634819PMC
September 2017
6 Reads

Quantification of mitochondrial DNA copy number in suspected cancer patients by a well optimized ddPCR method.

Biomol Detect Quantif 2017 Sep 31;13:32-39. Epub 2017 Aug 31.

Center for Primary Health Care Research, Lund University/Region Skåne, Malmö 20502, Sweden.

Changes in mitochondrial DNA (mtDNA) content is a useful clinical biomarker for various diseases, however results are controversial as several analytical factors can affect measurement of mtDNA. MtDNA is often quantified by taking ratio between a target mitochondrial gene and a reference nuclear gene (mtDNA/nDNA) using quantitative real time PCR often on two separate experiments. It measures relative levels by using external calibrator which may not be comparable across laboratories. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634817PMC
September 2017
7 Reads

*K-means and cluster models for cancer signatures.

Biomol Detect Quantif 2017 Sep 2;13:7-31. Epub 2017 Aug 2.

Centre for Computational Biology, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.

We present *K-means clustering algorithm and source code by expanding statistical clustering methods applied in https://ssrn.com/abstract=2802753 to quantitative finance. *K-means is statistically deterministic without specifying initial centers, etc. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634820PMC
September 2017
6 Reads

The Unyvero P55 'sample-in, answer-out' pneumonia assay: A performance evaluation.

Biomol Detect Quantif 2017 Sep 28;13:1-6. Epub 2017 Jun 28.

Centre for Clinical Microbiology, Division of Infection and Immunity, University College London, Royal Free Campus, Roland Hill Street, London, UK.

Background: O'Neill's recent Review on Antimicrobial Resistance expressed the view that by 2020 high-income countries should make it mandatory to support antimicrobial prescribing with rapid diagnostic evidence whenever possible.

Methods: Routine microbiology diagnosis of 95 respiratory specimens from patients with severe infection were compared with those generated by the Unyvero P55 test, which detects 20 pathogens and 19 antimicrobial resistance markers. Supplementary molecular testing for antimicrobial resistance genes, comprehensive culture methodology and 16S rRNA sequencing were performed. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634815PMC
September 2017
9 Reads

How to speed up the polymerase chain reaction.

Authors:
Stephen A Bustin

Biomol Detect Quantif 2017 Jun 20;12:10-14. Epub 2017 Jun 20.

Faculty of Medical Science, Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK.

Reducing the time taken to run qPCR assays on today's qPCR cyclers is rather straightforward and requires no specialised reagents or instruments. As the first article in a new series of short technical reports, I demonstrate that it is possible to reduce significantly both denaturation temperatures and cycling times, whilst retaining sensitivity and specificity of the original qPCR conditions. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496742PMC
June 2017
2 Reads

The continuing problem of poor transparency of reporting and use of inappropriate methods for RT-qPCR.

Authors:
Stephen Bustin

Biomol Detect Quantif 2017 Jun 23;12:7-9. Epub 2017 May 23.

Faculty of Medical Science, Anglia Ruskin University, Bishop Hall Lane, Chelmsford, CM1 1SQ, UK.

Attendance at this year's European Calcified Tissue Society's (ECTS) Congress reveals that the methods used to obtain qPCR results continue to be significantly flawed and that and their reporting remain inadequate. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496741PMC

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

Biomol Detect Quantif 2017 Jun 29;12:1-6. Epub 2017 Apr 29.

TATAA Biocenter AB, Gothenburg, Sweden.

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496743PMC
June 2017
103 Reads

Molecular techniques for the personalised management of patients with chronic myeloid leukaemia.

Biomol Detect Quantif 2017 Mar 14;11:4-20. Epub 2017 Feb 14.

Centre for Haematology, Department of Medicine, Imperial College London Hammersmith Hospital, London UK.

Chronic myeloid leukemia (CML) is the paradigm for targeted cancer therapy. RT-qPCR is the gold standard for monitoring response to tyrosine kinase-inhibitor (TKI) therapy based on the reduction of blood or bone marrow . Some patients with CML and very low or undetectable levels of transcripts can stop TKI-therapy without CML recurrence. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348117PMC
March 2017
25 Reads

Reproducibility of biomedical research - The importance of editorial vigilance.

Biomol Detect Quantif 2017 Mar 21;11:1-3. Epub 2017 Feb 21.

Analytical Microbiology, School of Bioscience and Medicine, Faculty of Health and Medical Science, University of Surrey, Guildford, GU2 7XH, United Kingdom.

Many journal editors are a failing to implement their own authors' instructions, resulting in the publication of many articles that do not meet basic standards of transparency, employ unsuitable data analysis methods and report overly optimistic conclusions. This problem is particularly acute where quantitative measurements are made and results in the publication of papers that lack scientific rigor and contributes to the concerns with regard to the reproducibility of biomedical research. This hampers research areas such as biomarker identification, as reproducing all but the most striking changes is challenging and translation to patient care rare. Read More

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http://dx.doi.org/10.1016/j.bdq.2017.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348116PMC
March 2017
2 Reads

Digital polymerase chain reaction for characterisation of DNA reference materials.

Biomol Detect Quantif 2016 Dec 3;10:47-49. Epub 2016 May 3.

National Measurement Institute, Lindfield, NSW 2070, Australia.

Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154631PMC
December 2016
1 Read

Three-color crystal digital PCR.

Biomol Detect Quantif 2016 Dec 3;10:34-46. Epub 2016 Nov 3.

Stilla Technologies, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France.

Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154636PMC
December 2016
5 Reads

Digital PCR dynamic range is approaching that of real-time quantitative PCR.

Biomol Detect Quantif 2016 Dec 2;10:31-33. Epub 2016 Nov 2.

Molecular and Cell Biology Team, LGC, Teddington, United Kingdom.

Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154633PMC
December 2016
1 Read

Designing and interpretation of digital assays: Concentration of target in the sample and in the source of sample.

Biomol Detect Quantif 2016 Dec 17;10:24-30. Epub 2016 May 17.

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, Warsaw 01-224, Poland; Curiosity Diagnostics Sp. z o.o. Kasprzaka 44/52, Warsaw 01-224, Poland.

We explain how to design classic digital assays, comprising identical partitions, in order to obtain the required precision of the estimate within a defined range of concentrations. The design, including the number and volume of partitions, depends significantly on whether the assay is to assess the concentration of the target analyte or (e.g. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S22147535163000
Publisher Site
http://dx.doi.org/10.1016/j.bdq.2016.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154637PMC
December 2016
2 Reads

Fundamentals of multiplexing with digital PCR.

Biomol Detect Quantif 2016 Dec 27;10:15-23. Epub 2016 May 27.

Digital Biology Centre, Bio-Rad Laboratories Inc., 5731 West Las Positas Boulevard, Pleasanton, CA 94588, United States.

Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154634PMC
December 2016
8 Reads

Applicability of digital PCR to the investigation of pediatric-onset genetic disorders.

Biomol Detect Quantif 2016 Dec 8;10:9-14. Epub 2016 Aug 8.

Center for Applied Clinical Genomics, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA; Center for Pediatric Research, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA; Department of Biological Sciences, University of Delaware, Newark, DE, USA; Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA, USA.

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. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154671PMC
December 2016

Homogeneous and digital proximity ligation assays for the detection of toxins A and B.

Biomol Detect Quantif 2016 Dec 31;10:2-8. Epub 2016 Aug 31.

Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, Essex, UK.

Background: 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. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S22147535163001
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http://dx.doi.org/10.1016/j.bdq.2016.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154635PMC
December 2016
6 Reads

Digital PCR, a technique for the future

Biomol Detect Quantif 2016 12 25;10. Epub 2016 Nov 25.

Molecular and Cell Biology, Science and Innovation, LGC, Queens Road, Teddington TW11 0LY, United Kingdom; School of Biosciences & Medicine, Faculty of Health & Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom.

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http://dx.doi.org/10.1016/j.bdq.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154632PMC
December 2016
3 Reads

Science in the UK - whereto now?

Authors:
Stephen Bustin

Biomol Detect Quantif 2016 Sep 21;9:A1-4. Epub 2016 Aug 21.

Postgraduate Medical Institute, Faculty of Medical Science, Anglia Ruskin University, Chelmsford, Essex, UK.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037121PMC
http://dx.doi.org/10.1016/j.bdq.2016.08.001DOI Listing
September 2016
1 Read

Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

Biomol Detect Quantif 2016 Sep 30;9:29-39. Epub 2016 Aug 30.

Directorate for Health, Consumers and Reference Materials, Joint Research Centre, European Commission, Retieseweg 111, 2440 Geel, Belgium.

Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007884PMC
September 2016
4 Reads

Influence of primer & probe chemistry and amplification target on reverse transcription digital PCR quantification of viral RNA.

Biomol Detect Quantif 2016 Sep 27;9:20-8. Epub 2016 Aug 27.

European Commission, Joint Research Centre, Retieseweg 111, B-2440 Geel, Belgium.

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 influenza A 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(®), were compared with two RNA template types: i) total genomic RNA extracted from cell cultured influenza A and ii) a synthetically prepared RNA transcript (In vitro transcribed RNA). Read More

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http://dx.doi.org/10.1016/j.bdq.2016.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007883PMC
September 2016
17 Reads

Methods for comparing multiple digital PCR experiments.

Biomol Detect Quantif 2016 Sep 10;9:14-9. Epub 2016 Aug 10.

University of Wroclaw, Faculty of Biotechnology, Department of Genomics, Wroclaw, Poland.

The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983647PMC
September 2016
6 Reads

Flexible analysis of digital PCR experiments using generalized linear mixed models.

Biomol Detect Quantif 2016 Sep 24;9:1-13. Epub 2016 Jun 24.

Department of Mathematical Modelling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Ghent, Belgium; National Institute for Applied Statistics Research Australia (NIASRA), University of Wollongong, NSW 2522, Australia.

The use of digital PCR for quantification of nucleic acids is rapidly growing. A major drawback remains the lack of flexible data analysis tools. Published analysis approaches are either tailored to specific problem settings or fail to take into account sources of variability. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983648PMC
September 2016
4 Reads

Diagnostic RAS mutation analysis by polymerase chain reaction (PCR).

Authors:
Ian A Cree

Biomol Detect Quantif 2016 Jun 6;8:29-32. Epub 2016 Jun 6.

Department of Pathology, University Hospitals Coventry and Warwickshire, Walsgrave, Coventry CV2 2DX, United Kingdom.

RAS mutation analysis is an important companion diagnostic test. Treatment of colorectal cancer with anti-Epidermal Growth Factor Receptor (EGFR) therapy requires demonstration of RAS mutation status (both KRAS and NRAS), and it is good practice to include BRAF. In Non-Small Cell Lung Cancer (NSCLC) and melanoma, assessment of RAS mutation status can be helpful in triaging patient samples for more extensive testing. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906127PMC
June 2016
2 Reads

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Biomol Detect Quantif 2016 Jun 6;8:15-28. Epub 2016 Jun 6.

LGC, Teddington, United Kingdom.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906133PMC
June 2016
28 Reads

Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs.

Biomol Detect Quantif 2016 Jun 8;8:9-14. Epub 2016 Apr 8.

Institute of Molecular Biology and Genetics, Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia.

Background: microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S22147535163000
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http://dx.doi.org/10.1016/j.bdq.2016.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906134PMC
June 2016
39 Reads

Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements.

Biomol Detect Quantif 2016 Jun 9;8:1-8. Epub 2016 Mar 9.

Bioassay Methods Group, Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, USA.

NIST standard reference material (SRM) 2373 was developed to improve the measurements of the HER2 gene amplification in DNA samples. SRM 2373 consists of genomic DNA extracted from five breast cancer cell lines with different amounts of amplification of the HER2 gene. The five components are derived from the human cell lines SK-BR-3, MDA-MB-231, MDA-MB-361, MDA-MB-453, and BT-474. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906140PMC
June 2016
3 Reads

Reliable measurement of multiplexed biomarker panel of RNA transcripts.

Authors:
Robert Wielgosz

Biomol Detect Quantif 2016 Jun 6;8:A1. Epub 2016 Jun 6.

Bureau International des Poids et Mesures, Pavillon de Breteuil, F-92312, Sevres Cedex, France.

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http://dx.doi.org/10.1016/j.bdq.2016.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328640PMC

Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

Biomol Detect Quantif 2016 Mar 19;7:27-33. Epub 2016 Feb 19.

Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD, USA.

Aims: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR).

Methods And Results: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827694PMC
March 2016
4 Reads

qPCR based mRNA quality score show intact mRNA after heat stabilization.

Biomol Detect Quantif 2016 Mar 10;7:21-6. Epub 2016 Feb 10.

Denator AB, Uppsala, Sweden.

Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. Read More

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http://dx.doi.org/10.1016/j.bdq.2016.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827693PMC
March 2016
3 Reads

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Biomol Detect Quantif 2016 Mar 7;7:9-20. Epub 2016 Jan 7.

Bavarian Health and Food Safety Authority (LGL), Veterinaerstr. 2, 85764 Oberschleissheim, Germany.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. Read More

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http://dx.doi.org/10.1016/j.bdq.2015.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827695PMC
March 2016
74 Reads