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Digital PCR

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https://www.readbyqxmd.com/read/28448765/digital-assays-part-i-partitioning-statistics-and-digital-pcr
#1
Amar S Basu
A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error...
April 1, 2017: SLAS Technology
https://www.readbyqxmd.com/read/28447422/minimal-residual-disease-in-acute-myelogenous-leukemia
#2
REVIEW
N M Cruz, N Mencia-Trinchant, D C Hassane, M L Guzman
Treatment of acute myelogenous leukemia (AML) over the past four decades remains mostly unchanged and the prognosis for the majority of patients remains poor. Most of the significant advances that have been observed are in defining cytogenetic abnormalities, as well as the genetic and epigenetic profiles of AML patients. While new cytogenetic and genetic aberrations such as the FLT3-ITD and NPM1 mutations are able to guide prognosis for the majority of patients with AML, outcomes are still dismal and relapse rates remain high...
May 2017: International Journal of Laboratory Hematology
https://www.readbyqxmd.com/read/28446770/instability-of-8e5-calibration-standard-revealed-by-digital-pcr-risks-inaccurate-quantification-of-hiv-dna-in-clinical-samples-by-qpcr
#3
Eloise Busby, Alexandra S Whale, R Bridget Ferns, Paul R Grant, Gary Morley, Jonathan Campbell, Carole A Foy, Eleni Nastouli, Jim F Huggett, Jeremy A Garson
Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides 'absolute' quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples...
April 26, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28445137/monitoring-of-kras-mutated-ctdna-to-discriminate-pseudo-progression-from-true-progression-during-anti-pd-1-treatment-of-lung-adenocarcinoma
#4
Nicolas Guibert, Julien Mazieres, Myriam Delaunay, Anne Casanova, Magali Farella, Laura Keller, Gilles Favre, Anne Pradines
OBJECTIVES: Pseudo-progression is a rare but worrying situation for both clinicians and patients during immunotherapy. Dedicated ir-RECIST criteria have been established to improve this situation. However, this can be sometimes considered inadequate and patients experiencing true progression may then receive inefficient treatments. Additional reliable tools to discriminate pseudo from true progression are thus needed. So far, no biomarker has been identified to distinguish pseudo from true progression...
April 7, 2017: Oncotarget
https://www.readbyqxmd.com/read/28444752/prevalence-and-pathogen-load-of-campylobacter-spp-salmonella-enterica-and-escherichia-coli-o157-o145-serogroup-in-sheep-faeces-collected-at-sale-yards-and-in-abattoir-effluent-in-western-australia
#5
R Yang, S Abraham, G E Gardner, U Ryan, C Jacobson
OBJECTIVE: Develop a multiplex quantitative PCR assay to investigate the prevalence and shedding of Escherichia coli O157/O145, Salmonella spp. and Campylobacter spp. in sheep at sale yards and abattoirs. METHODS: A qPCR for E. coli O157/O145 was developed, validated and multiplexed with an existing qPCR for Campylobacter and Salmonella enterica. The absolute numbers of E. coli O157/O145, Campylobacter and Salmonella in control samples was determined using droplet digital PCR...
May 2017: Australian Veterinary Journal
https://www.readbyqxmd.com/read/28444728/circulating-cell-free-braf-v600e-as-a-biomarker-in-children-with-langerhans-cell-histiocytosis
#6
Sébastien Héritier, Zofia Hélias-Rodzewicz, Hélène Lapillonne, Nathalie Terrones, Sonia Garrigou, Corinne Normand, Mohamed-Aziz Barkaoui, Jean Miron, Geneviève Plat, Nathalie Aladjidi, Anne Pagnier, Anne Deville, Marion Gillibert-Yvert, Despina Moshous, Alain Lefèvre-Utile, Anne Lutun, Catherine Paillard, Caroline Thomas, Eric Jeziorski, Philippe Nizard, Valérie Taly, Jean-François Emile, Jean Donadieu
The BRAF(V600E) mutation is reported in half of patients with Langerhans cell histiocytosis (LCH). This study investigated the detection of the BRAF(V600E) allele in circulating cell-free (ccf) DNA in a paediatric LCH cohort. Children with BRAF(V600E) -mutated LCH were investigated to detect ccf BRAF(V600E) at diagnosis (n = 48) and during follow-up (n = 17) using a picolitre-droplet digital PCR assay. At diagnosis, ccf BRAF(V600E) was positive in 15/15 (100%) patients with risk-organ positive multisystem (RO+ MS) LCH, 5/12 (42%) of patients with RO- MS LCH and 3/21 (14%) patients with single-system (SS) LCH (P < 0·001, Fisher's exact test)...
April 25, 2017: British Journal of Haematology
https://www.readbyqxmd.com/read/28443110/expression-profiling-of-castanea-genes-during-resistant-and-susceptible-interactions-with-the-oomycete-pathogen-phytophthora-cinnamomi-reveal-possible-mechanisms-of-immunity
#7
Carmen Santos, Sofia Duarte, Sara Tedesco, Pedro Fevereiro, Rita L Costa
The most dangerous pathogen affecting the production of chestnuts is Phytophthora cinnamomi a hemibiotrophic that causes root rot, also known as ink disease. Little information has been acquired in chestnut on the molecular defense strategies against this pathogen. The expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight candidate genes displayed differentially expressed levels depending on genotype and time-point after inoculation...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28442757/comparison-of-circulating-tumour-cells-and-circulating-cell-free-epstein-barr-virus-dna-in-patients-with-nasopharyngeal-carcinoma-undergoing-radiotherapy
#8
Jess Honganh Vo, Wen Long Nei, Min Hu, Wai Min Phyo, Fuqiang Wang, Kam Weng Fong, Terence Tan, Yoke Lim Soong, Shie Lee Cheah, Kiattisa Sommat, Huiyu Low, Belinda Ling, Johnson Ng, Wan Loo Tan, Kian Sing Chan, Lynette Oon, Jackie Y Ying, Min-Han Tan
Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs). Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed...
December 19, 2016: Scientific Reports
https://www.readbyqxmd.com/read/28442288/expression-and-differential-regulation-of-hla-g-isoforms-in-the-retinal-pigment-epithelial-cell-line-arpe-19
#9
Signe Goul Svendsen, Maja Søberg Udsen, Marina Daouya, Tina Funck, Ching-Lien Wu, Edgardo D Carosella, Joël LeMaoult, Thomas Vauvert F Hviid, Carsten Faber, Mogens Holst Nissen
The purpose of this study was to examine if HLA-G is expressed in the retinal pigment epithelium (RPE) cells of the eye. The RPE comprises the outer most layer of the retina and as such defines the interface to the blood and contributes to the immune privilege in the posterior part of the eye. One way the RPE might be regulating the immune system could be by expressing the non-classical human leukocyte antigen (HLA) molecule, HLA-G. We therefore sought to define if the RPE cell line, ARPE-19, expressed HLA-G and analyse the regulation as a response to pro-inflammatory cytokines...
April 22, 2017: Human Immunology
https://www.readbyqxmd.com/read/28441604/digital-quantitative-analysis-of-microrna-in-single-cell-based-on-ligation-depended-polymerase-colony-polony
#10
Hui Wang, Honghong Wang, Xinrui Duan, Chenghui Liu, Zhengping Li
The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template...
April 7, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28441386/graft-derived-cell-free-dna-a-noninvasive-early-rejection-and-graft-damage-marker-in-liver-transplantation-a-prospective-observational-multicenter-cohort-study
#11
Ekkehard Schütz, Anna Fischer, Julia Beck, Markus Harden, Martina Koch, Tilo Wuensch, Martin Stockmann, Björn Nashan, Otto Kollmar, Johannes Matthaei, Philipp Kanzow, Philip D Walson, Jürgen Brockmöller, Michael Oellerich
BACKGROUND: Graft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection...
April 2017: PLoS Medicine
https://www.readbyqxmd.com/read/28439837/detection-of-micrornas-using-chip-based-quantstudio-3d-digital-pcr
#12
Cristina Borzi, Linda Calzolari, Davide Conte, Gabriella Sozzi, Orazio Fortunato
Digital PCR (dPCR) is an innovative approach for detection and quantification of nucleic acid that offers an alternative method to conventional real-time quantitative PCR for absolute quantification. dPCR is a highly precise and sensitive technique that does not require a standard reference, making it a suitable method for the detection of microRNAs. The potential of these small noncoding RNA as biomarkers is on the rise, especially due to their presence in body fluids, making them easily accessible. Nevertheless, the problem of lack of consensus regarding an optimal method for miRNAs normalization compromises their use...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28437435/infective-respiratory-syncytial-virus-is-present-in-human-cord-blood-samples-and-most-prevalent-during-winter-months
#13
Angela Mary Fonceca, Abha Chopra, Avram Levy, Paul Stanton Noakes, Matthew Wee-Peng Poh, Natasha Leanne Bear, Susan Prescott, Mark Lloyd Everard
BACKGROUND: Human respiratory syncytial virus (RSV) remains the most common cause of severe lower respiratory tract disease amongst infants, and continues to cause annual epidemics of respiratory disease every winter worldwide. Demonstrating placental transmission of viable RSV in human samples is a major paradigm shift in respiratory routes considered likely for RSV transmission. METHODS: Droplet digital PCR (ddPCR) was used to identify RSV present in cord blood mononucleocytes (CBM)...
2017: PloS One
https://www.readbyqxmd.com/read/28437026/analyzing-epidermal-growth-factor-receptor-mutation-status-changes-in-advanced-non-small-cell-lung-cancer-at-different-sampling-time-points-of-blood-within-one%C3%A2-day
#14
Jin Wang, Hua Bai, Chaoyu Hong, Jie Wang, Tong-Hua Mei
BACKGROUND: We investigated whether different sampling time-points within one day would influence epidermal growth factor receptor mutation (EGFRm) status in plasma and evaluated the clinical outcomes according to the quantity analysis of EGFRm in circulating tumor DNA (ctDNA) in non-small-cell lung cancer (NSCLC). METHODS: EGFR-tyrosine kinase inhibitor naïve advanced NSCLC patients who carried EGFRm in both tissues and ctDNA were enrolled in this study. Plasma samples were collected at three time-points within one day (at 8 am, 11 am and 2 pm) for EGFRm analysis by droplet digital PCR...
April 24, 2017: Thoracic Cancer
https://www.readbyqxmd.com/read/28426683/genome-wide-identification-and-characterization-of-mirnaome-from-tomato-solanum-lycopersicum-roots-and-root-knot-nematode-meloidogyne-incognita-during-susceptible-interaction
#15
Pritam Kaur, Neha Shukla, Gopal Joshi, Cheeni VijayaKumar, Arun Jagannath, Manu Agarwal, Shailendra Goel, Amar Kumar
Root-knot nematodes (RKNs, Meloidogyne spp.) are the most damaging plant parasites causing severe losses to crop production. The present study reports genome-wide identification and characterization of both tomato and RKN miRNAs simultaneously from RKN-infected susceptible tomato roots using high-throughput sequencing technique. RNAseq data from 11 small RNA libraries derived from 5 disease development stages identified 281 novel miRNAs of tomato in addition to 52 conserved and 4 variants of conserved miRNAs...
2017: PloS One
https://www.readbyqxmd.com/read/28424530/molecular-diagnostic-assays-based-on-cpn60-ut-sequences-reveal-the-geographic-distribution-of-subgroup-16srxiii-a-i-i-phytoplasma-in-mexico
#16
Edel Pérez-López, Douglas Rodríguez-Martínez, Chrystel Y Olivier, Mauricio Luna-Rodríguez, Tim J Dumonceaux
Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma...
April 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28423028/four-human-plasmodium-species-quantification-using-droplet-digital-pcr
#17
Suttipat Srisutham, Naowarat Saralamba, Benoit Malleret, Laurent Rénia, Arjen M Dondorp, Mallika Imwong
Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection...
2017: PloS One
https://www.readbyqxmd.com/read/28422522/molecular-genetic-characterization-of-a-chinese-family-with-severe-split-hand-foot-malformation
#18
Lihua Cao, Wei Yang, Shusen Wang, Chen Chen, Xue Zhang, Yang Luo
AIMS: Split hand/foot malformation (SHFM) is a congenital limb malformation characterized by underdeveloped or absent central digital rays, clefts of the hands and feet, and variable syndactyly of the remaining digits. SHFM is a genetically heterogeneous disease; the aim of this study was to identify pathogenic variations in a Chinese family with SHFM. MATERIALS AND METHODS: Haplotype analyses with microsatellite markers covering the five SHFM loci were performed to localize the causative locus...
April 19, 2017: Genetic Testing and Molecular Biomarkers
https://www.readbyqxmd.com/read/28422403/blood-chimerism-in-dizygotic-monochorionic-twins-during-five-years-observation
#19
Morten Hanefeld Dziegiel, Morten Høgh Hansen, Sofie Haedersdal, Angela Natalie Barrett, Klaus Rieneck, Katharina Maria Main, Anne Todsen Hansen, Frederik Banch Clausen
Dizygotic monochorionic twin pregnancies can result in blood chimerism due to in utero twin-to-twin exchange of stem cells. In this case, we examined the proportion of allogeneic red blood cells (RBCs) by flow cytometry and the proportion of allogeneic nucleated cells by digital PCR at seven months and again at five years. We found an increase in the proportion of allogeneic cells from 63% to 89% in one twin, and a similar increase in autologous cells in the other twin from 57% to 84%. A paradigm for stem cell therapy could be modelled on this case: induction of tolerance and chimerism by antenatal transfusion of donor stem cells...
April 19, 2017: American Journal of Transplantation
https://www.readbyqxmd.com/read/28419670/sglt2-and-sglt1-renal-expression-in-patients-with-type-2-diabetes
#20
Anna Solini, Chiara Rossi, Chiara Maria Mazzanti, Agnese Proietti, Hermann Koepsell, Ele Ferrannini
AIMS: The notion of an increased expression of SGLT2 in the kidney of patients with type 2 diabetes (T2DM) is based on a single ex vivo study of tubular cells harvested from the urine. DESIGN AND METHODS: We measured SGLT2 and SGLT1 expression in unaffected renal tissue from 19 T2DM patients and 20 age- and eGFR-matched nondiabetic subjects (CT) undergoing unilateral nephrectomy. Expression of SGLT2 and SGLT1 - and their cognate basolateral transporters GLUT2 and GLUT1 - was quantified by real-time and digital PCR; an affinity-purified antibody against human SGLT2 was used localize SGLT2 by immunohistochemistry...
April 17, 2017: Diabetes, Obesity & Metabolism
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