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Digital PCR

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https://www.readbyqxmd.com/read/28350455/model-based-classification-for-digital-pcr-your-umbrella-for-rain
#1
Bart K M Jacobs, Els Goetghebeur, Jo Vandesompele, Ariane De Ganck, Nele Nijs, Anneleen Beckers, Nina Papazova, Nancy Hélène C J Roosens, Lieven Clement
Standard data analysis pipelines for digital PCR estimate the concentration of a target nucleic acid by digitizing the end-point fluorescence of the parallel micro-PCR reactions using an automated hard threshold. While it is known that misclassification has a major impact on the concentration estimate and substantially reduces accuracy, the uncertainty of this classification is typically ignored. We introduce a model-based clustering method to estimate the probability that the target is present (absent) in a partition conditional on its observed fluorescence and the distributional shape in no template control samples...
March 28, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28339648/three-dimensional-testicular-organoid-a-novel-tool-for-the-study-of-human-spermatogenesis-and-gonadotoxicity-in-vitro%C3%A2
#2
Samuel S Pendergraft, Hooman Sadri-Ardekani, Anthony Atala, Colin E Bishop
Existing methods for evaluating the potential gonadotoxicity of environmental agents and pharmaceutical compounds rely heavily on animal studies. The current gold standard in vivo functional assays in animals are limited in their human predictive capacity. In addition, existing human two-dimensional in vitro models of testicular toxicity do not accurately reflect the in vivo situation. A more reliable testicular in vitro model system is needed to better assess the gonadotoxic potential of drugs prior to progression into clinical trials...
February 17, 2017: Biology of Reproduction
https://www.readbyqxmd.com/read/28333573/new-approaches-for-characterization-of-the-genetic-stability-of-vaccine-cell-lines
#3
Siemon Ng, Lucy Gisonni-Lex, Ali Azizi
The genetic stability of cell lines is a critical analytical attribute required to demonstrate the quality of cells over time. During cell passage, mutations can arise in the genomic DNA, potentially leading to changes in the final vaccine product. The identity and integrity of master cell banks, extended cell banks, complementing cell lines or recombinant cell lines expressing transgenes has to be tested throughout the production process by the vaccine manufacturer. Over the past few years, the traditional methods for evaluation of genetic stability have been replaced with molecular approaches including quantitative PCR, digital PCR and high throughput sequencing...
February 23, 2017: Human Vaccines & Immunotherapeutics
https://www.readbyqxmd.com/read/28330559/dge-seq-analysis-of-mur3-related-arabidopsis-mutants-provides-insight-into-how-dysfunctional-xyloglucan-affects-cell-elongation
#4
Zongchang Xu, Meng Wang, Dachuan Shi, Gongke Zhou, Tiantian Niu, Michael G Hahn, Malcolm A O'Neill, Yingzhen Kong
Our previous study of the Arabidopsis mur3-3 mutant and mutant plants in which the mur3-3 phenotypes are suppressed (xxt2mur3-3, xxt5mur3-3, xxt1xxt2mur3-3 and 35Spro:XLT2:mur3-3) showed that hypocotyl cell elongation is decreased in plants that synthesize galactose-deficient xyloglucan. To obtain genome-wide insight into the transcriptome changes and regulatory networks that may be involved in this decreased elongation, we performed digital gene expression analyses of the etiolated hypocotyls of wild type (WT), mur3-3 and the four suppressor lines...
May 2017: Plant Science: An International Journal of Experimental Plant Biology
https://www.readbyqxmd.com/read/28330468/tumor-burden-monitoring-using-cell-free-tumor-dna-could-be-limited-by-tumor-heterogeneity-in-advanced-breast-cancer-and-should-be-evaluated-together-with-radiographic-imaging
#5
José Angel García-Saenz, Patricia Ayllón, Marion Laig, Daniel Acosta-Eyzaguirre, Marta García-Esquinas, Myriam Montes, Julián Sanz, Miguel Barquín, Fernando Moreno, Vanesa Garcia-Barberan, Eduardo Díaz-Rubio, Trinidad Caldes, Atocha Romero
BACKGROUND: Accurate measurement of tumor burden in breast cancer disease is essential to improve the clinical management of patients. In this study, we evaluate whether the fluctuations in the fraction of PIK3CA mutant allele correlates with tumor response according to RECIST criteria and tumor markers quantification. METHODS: Eighty six plasma samples were analyzed by digital PCR using Rare Mutation Assays for E542K, E545K and H1047R. Mutant cfDNA and tumor markers CA15-3 and CEA were compared with radiographic imaging...
March 22, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28327545/calibration-free-assays-on-standard-real-time-pcr-devices
#6
Pawel R Debski, Kamil Gewartowski, Seweryn Bajer, Piotr Garstecki
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...
March 22, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28322802/simple-quantitative-pcr-analysis-for-allelic-pten-loss-in-tumor-progression
#7
Xingping Wu, Ailin Zhu, Xiaowu Pang, Guiqin Xie, Xinbin Gu
We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR...
March 18, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28322208/prevalence-and-pathogen-load-estimates-for-the-fungus-batrachochytrium-dendrobatidis-are-impacted-by-its-dna-copy-number-variation
#8
Eria A Rebollar, Douglas C Woodhams, Brandon LaBumbard, Jos Kielgast, Reid N Harris
The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibians and white nose syndrome in bats. Chytridiomycosis is caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and is responsible for declines and extinctions of amphibians worldwide...
March 21, 2017: Diseases of Aquatic Organisms
https://www.readbyqxmd.com/read/28319152/a-digital-pcr-method-for-identifying-and-quantifying-adulteration-of-meat-species-in-raw-and-processed-food
#9
Junan Ren, Tingting Deng, Wensheng Huang, Ying Chen, Yiqiang Ge
Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination...
2017: PloS One
https://www.readbyqxmd.com/read/28318500/mutations-in-tmem260-cause-a-pediatric-neurodevelopmental-cardiac-and-renal-syndrome
#10
Asaf Ta-Shma, Tahir N Khan, Asaf Vivante, Jason R Willer, Pavle Matak, Chaim Jalas, Ben Pode-Shakked, Yishay Salem, Yair Anikster, Friedhelm Hildebrandt, Nicholas Katsanis, Orly Elpeleg, Erica E Davis
Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon...
March 11, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28303131/multiplexed-single-intact-cell-droplet-digital-pcr-music-ddpcr-method-for-specific-detection-of-enterohemorrhagic-e-coli-ehec-in-food-enrichment-cultures
#11
Tanis C McMahon, Burton W Blais, Alex Wong, Catherine D Carrillo
Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28303130/new-approaches-on-quantification-of-campylobacter-jejuni-in-poultry-samples-the-use-of-digital-pcr-and-real-time-pcr-against-the-iso-standard-plate-count-method
#12
Bojan Papić, Mateja Pate, Urška Henigman, Urška Zajc, Igor Gruntar, Majda Biasizzo, Matjaž Ocepek, Darja Kušar
Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28300664/gene-expression-changes-in-immune-response-pathways-following-oral-administration-of-tetrabromobisphenol-a-tbbpa-in-female-wistar-han-rats
#13
Samantha M Hall, Sherry J Coulter, Gabriel A Knudsen, J Michael Sanders, Linda S Birnbaum
Tetrabromobisphenol A (TBBPA) is a brominated flame retardant used globally at high volumes, primarily in the epoxy resin of circuit boards. It has been detected in the environment and in humans. The National Toxicology Program found that chronic oral TBBPA treatment of 250mg/kg and higher caused an increased incidence of uterine lesions in female Wistar Han rats. The present laboratory has previously reported changes in gene expression associated with estrogen homeostasis in liver and uterine tissue of adult female Wistar Han rats after five days of gavage with 250mg/kg of TBBPA...
March 11, 2017: Toxicology Letters
https://www.readbyqxmd.com/read/28298452/applications-of-digital-pcr-for-clinical-microbiology
#14
Jane Kuypers, Keith R Jerome
Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory...
March 15, 2017: Journal of Clinical Microbiology
https://www.readbyqxmd.com/read/28294696/cerebrospinal-fluid-mtdna-concentration-is-elevated-in-multiple-sclerosis-disease-and-responds-to-treatment
#15
Cyra E Leurs, Petar Podlesniy, Ramon Trullas, Lisanne Balk, Martijn D Steenwijk, Arjan Malekzadeh, Fredrik Piehl, Bernard Mj Uitdehaag, Joep Killestein, Jack van Horssen, C E Teunissen
BACKGROUND: Mitochondrial dysfunction is increasingly recognized as an important feature of multiple sclerosis (MS) pathology and may be relevant for clinical disease progression. However, it is unknown whether mitochondrial DNA (mtDNA) levels in the cerebrospinal fluid (CSF) associate with disease progression and therapeutic response. OBJECTIVES: To evaluate whether CSF concentrations of mtDNA in MS patients can serve as a marker of ongoing neuropathology and may be helpful to differentiate between MS disease subtypes...
March 1, 2017: Multiple Sclerosis: Clinical and Laboratory Research
https://www.readbyqxmd.com/read/28292978/validating-a-fully-automated-real-time-pcr-based-system-for-use-in-the-molecular-diagnostic-analysis-of-colorectal-carcinoma-a-comparison-with-ngs-and-ihc
#16
Richard Colling, Lai Mun Wang, Elizabeth Soilleux
BACKGROUND: Molecular testing is increasingly needed in colorectal carcinoma (CRC) and the current clinically relevant mutations are in BRAF, KRAS and NRAS. This study aimed to further validate a new alternative polymerase chain reaction (PCR) platform (Idylla, Biocartis) against existing next-generation sequencing (NGS) and immunohistochemistry (IHC) assays. METHODS: 56 Idylla tests were performed on 43 CRC cases, in a total of 74 comparisons against an NGS panel (Ion Torrent) and the VE1 (anti-BRAF) antibody IHC...
March 14, 2017: Journal of Clinical Pathology
https://www.readbyqxmd.com/read/28288812/preparation-of-ms2-based-nanoparticles-as-control-and-standard-materials-for-the-molecular-detection-of-dengue-virus-serotypes
#17
Yu Fu, Guojing Wang, Qisheng Wu, Xin Yang, Rui Zhang, Kuo Zhang, Guigao Lin, Yanxi Han, Lihua Bao, Ziyang Li, Jinming Li
To quantify dengue virus (DENV) and evaluate the performance of clinical laboratories using quantitative real-time polymerase chain reaction (qRT-PCR) assays, we constructed high-efficiency expression systems to produce DENV-1 to 4 nanoparticles and assessed their suitability as standard and control materials in 20 laboratories across China. Targeted gene sequences of DENV-1 to 4 were synthesized and inserted into pACYC-Duet 1-MS2 recombinant plasmids to generate corresponding nanoparticle expression systems...
March 10, 2017: Virus Research
https://www.readbyqxmd.com/read/28285688/plasma-epidermal-growth-factor-receptor-mutation-testing-with-a-chip-based-digital-pcr-system-in-patients-with-advanced-non-small-cell-lung-cancer
#18
Norimitsu Kasahara, Hirotsugu Kenmotsu, Masakuni Serizawa, Rina Umehara, Akira Ono, Yasushi Hisamatsu, Kazushige Wakuda, Shota Omori, Kazuhisa Nakashima, Tetsuhiko Taira, Tateaki Naito, Haruyasu Murakami, Yasuhiro Koh, Keita Mori, Masahiro Endo, Takashi Nakajima, Masanobu Yamada, Masatoshi Kusuhara, Toshiaki Takahashi
OBJECTIVES: Epidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20)...
April 2017: Lung Cancer: Journal of the International Association for the Study of Lung Cancer
https://www.readbyqxmd.com/read/28284873/establishing-a-reference-dataset-for-the-authentication-of-spinal-muscular-atrophy-cell-lines-using-str-profiling-and-digital-pcr
#19
Deborah L Stabley, Jennifer Holbrook, Ashlee W Harris, Kathryn J Swoboda, Thomas O Crawford, Katia Sol-Church, Matthew E R Butchbach
Fibroblasts and lymphoblastoid cell lines (LCLs) derived from individuals with spinal muscular atrophy (SMA) have been and continue to be essential for translational SMA research. Authentication of cell lines helps ensure reproducibility and rigor in biomedical research. This quality control measure identifies mislabeling or cross-contamination of cell lines and prevents misinterpretation of data. Unfortunately, authentication of SMA cell lines used in various studies has not been possible because of a lack of a reference...
February 6, 2017: Neuromuscular Disorders: NMD
https://www.readbyqxmd.com/read/28283903/detection-of-esr1-mutations-in-plasma-and-tumors-from-metastatic-breast-cancer-patients-using-next-generation-sequencing
#20
Takehiro Yanagawa, Naofumi Kagara, Tomohiro Miyake, Tomonori Tanei, Yasuto Naoi, Masafumi Shimoda, Kenzo Shimazu, Seung Jin Kim, Shinzaburo Noguchi
PURPOSE: Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations. METHODS: Whole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients...
March 10, 2017: Breast Cancer Research and Treatment
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