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https://www.readbyqxmd.com/read/28928368/detection-of-clinically-relevant-copy-number-alterations-in-oral-cancer-progression-using-multiplexed-droplet-digital-pcr
#1
Curtis B Hughesman, X J David Lu, Kelly Y P Liu, Yuqi Zhu, Rebecca M Towle, Charles Haynes, Catherine F Poh
Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays...
September 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28923315/development-of-a-digital-droplet-pcr-assay-to-measure-hbv-dna-in-patients-receiving-long-term-tdf-treatment
#2
Yang Liu, Andrea L Cathcart, William E Delaney, Kathryn M Kitrinos
The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (<58 copies/mL) or target detected (TD) (between 58 to 169 copies/mL). Here we have developed a more sensitive digital droplet PCR (ddPCR) assay to evaluate the impact of long-term tenofovir disoproxil fumarate (TDF) treatment in patients that did or did not achieve HBsAg seroconversion. A ddPCR assay was developed to detect HBV DNA to 8 copies/mL...
September 15, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28921562/a-novel-approach-using-long-read-sequencing-and-ddpcr-to-investigate-gonadal-mosaicism-and-estimate-recurrence-risk-in-two-families-with-developmental-disorders
#3
Maria Wilbe, Sanna Gudmundsson, Josefin Johansson, Adam Ameur, Eva-Lena Stattin, Göran Annerén, Helena Malmgren, Carina Frykholm, Marie-Louise Bondeson
OBJECTIVE: De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo there is a recurrence risk due to parental germ line mosaicism, depending on the gonadal generation the mutation occurred. METHODS: We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction...
September 16, 2017: Prenatal Diagnosis
https://www.readbyqxmd.com/read/28910375/application-of-droplet-digital-pcr-for-quantitative-detection-of-spiroplasma-citri-in-comparison-with-real-time-pcr
#4
Yogita Maheshwari, Vijayanandraj Selvaraj, Subhas Hajeri, Raymond Yokomi
Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S...
2017: PloS One
https://www.readbyqxmd.com/read/28905136/highly-sensitive-detection-of-esr1-mutations-in-cell-free-dna-from-patients-with-metastatic-breast-cancer-using-molecular-barcode-sequencing
#5
Nanae Masunaga, Naofumi Kagara, Daisuke Motooka, Shota Nakamura, Tomohiro Miyake, Tomonori Tanei, Yasuto Naoi, Masafumi Shimoda, Kenzo Shimazu, Seung Jin Kim, Shinzaburo Noguchi
PURPOSE: We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). METHODS: The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR)...
September 13, 2017: Breast Cancer Research and Treatment
https://www.readbyqxmd.com/read/28901323/human-epidermal-growth-factor-receptor-2-amplification-detection-by-droplet-digital-polymerase-chain-reaction-in-formalin-fixed-paraffin-embedded-breast-and-gastric-cancer-samples
#6
Xingwen Wang, Yunyan Wu, Xueling Song, Chengtao Sun, Changshun Wu, Hong Feng
OBJECTIVE: Human epidermal growth factor receptor 2 (HER2) is an important biomarker for the precise individualized treatment including trastuzumab of HER2-positive breast and gastric cancer. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the routine analyses for formalin-fixed paraffin-embedded (FFPE) samples. However, IHC is variable and depends on the evaluator, and FISH is a labor intensive and expensive method. We evaluated the feasibility of droplet digital polymerase chain reaction (ddPCR) as a precise and quantitative method for HER2 amplification test...
2017: Journal of Cancer Research and Therapeutics
https://www.readbyqxmd.com/read/28900844/a-pilot-study-on-the-use-of-cerebrospinal-fluid-cell-free-dna-in-intramedullary-spinal-ependymoma
#7
Ian David Connolly, Yingmei Li, Wenying Pan, Eli Johnson, Linya You, Hannes Vogel, John Ratliff, Melanie Hayden Gephart
Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord...
September 12, 2017: Journal of Neuro-oncology
https://www.readbyqxmd.com/read/28895730/rheostatic-control-of-cas9-mediated-dna-double-strand-break-dsb-generation-and-genome-editing
#8
John C Rose, Jason J Stephany, Cindy T Wei, Douglas M Fowler, Dustin J Maly
We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels...
September 15, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28878842/efficacy-of-ezh2-inhibitory-drugs-in-human-papillomavirus-positive-and-human-papillomavirus-negative-oropharyngeal-squamous-cell-carcinomas
#9
Cameron D Lindsay, Morris A Kostiuk, Jeff Harris, Daniel A O'Connell, Hadi Seikaly, Vincent L Biron
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with rates of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) dramatically increasing. The overexpression of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase responsible for the trimethylation at lysine 27 of histone 3 (H3K27me3), is associated with a poor clinical prognosis and aggressive HPV-positive phenotypes. METHODS: We utilized three EZH2 pathway inhibitors, GSK-343, DZNeP, and EPZ-5687, and tested their efficacy in two HPV-positive and two HPV-negative OPSCC cell lines...
2017: Clinical Epigenetics
https://www.readbyqxmd.com/read/28868565/comparison-of-the-amplification-refractory-mutation-system-super-amplification-refractory-mutation-system-and-droplet-digital-pcr-for-t790%C3%A2-m-mutation-detection-in-non-small-cell-lung-cancer-after-failure-of-tyrosine-kinase-inhibitor-treatment
#10
Lucheng Zhu, Shirong Zhang, Yanping Xun, Yanping Jiang, Bing Xia, Xueqin Chen, Limin Wang, Hong Jiang, Shenglin Ma
Plasma mutation detection has the advantages of non-invasiveness and accessibility. Here, we evaluated three methods, the amplification refractory mutation system (ARMS), second-generation ARMS (SuperARMS), and droplet digital PCR (ddPCR), to assess their concordance and feasibility for the detection of mutations in plasma samples. Non-small lung cancer patients with stage IIIB/IV that were resistant to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) treatment were enrolled. Blood samples were collected within 14 days after TKI resistance...
September 3, 2017: Pathology Oncology Research: POR
https://www.readbyqxmd.com/read/28826609/a-droplet-digital-pcr-method-for-severe-combined-immunodeficiency-newborn-screening
#11
Noemi Vidal-Folch, Dragana Milosevic, Ramanath Majumdar, Dimitar Gavrilov, Dietrich Matern, Kimiyo Raymond, Piero Rinaldo, Silvia Tortorelli, Roshini S Abraham, Devin Oglesbee
Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch...
September 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28819142/development-and-inter-laboratory-assessment-of-droplet-digital-pcr-assays-for-multiplex-quantification-of-15-genetically-modified-soybean-lines
#12
Alexandra Bogožalec Košir, Bjørn Spilsberg, Arne Holst-Jensen, Jana Žel, David Dobnik
Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR)...
August 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28807002/high-resolution-measurement-of-duf1220-domain-copy-number-from-whole-genome-sequence-data
#13
David P Astling, Ilea E Heft, Kenneth L Jones, James M Sikela
BACKGROUND: DUF1220 protein domains found primarily in Neuroblastoma BreakPoint Family (NBPF) genes show the greatest human lineage-specific increase in copy number of any coding region in the genome. There are 302 haploid copies of DUF1220 in hg38 (~160 of which are human-specific) and the majority of these can be divided into 6 different subtypes (referred to as clades). Copy number changes of specific DUF1220 clades have been associated in a dose-dependent manner with brain size variation (both evolutionarily and within the human population), cognitive aptitude, autism severity, and schizophrenia severity...
August 14, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28802157/droplet-digital-pcr-for-rapid-enumeration-of-viral-genomes-and-particles-from-cells-and-animals-infected-with-orthopoxviruses
#14
Jeffrey L Americo, Patricia L Earl, Bernard Moss
Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer...
August 9, 2017: Virology
https://www.readbyqxmd.com/read/28796824/droplet-digital-polymerase-chain-reaction-ddpcr-assays-integrated-with-an-internal-control-for-quantification-of-bovine-porcine-chicken-and-turkey-species-in-food-and-feed
#15
Hanan R Shehata, Jiping Li, Shu Chen, Helen Redda, Shumei Cheng, Nicole Tabujara, Honghong Li, Keith Warriner, Robert Hanner
Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures...
2017: PloS One
https://www.readbyqxmd.com/read/28780426/characterization-of-oseltamivir-resistant-influenza-virus-populations-in-immunosuppressed-patients-using-digital-droplet-pcr-comparison-with-qpcr-and-next-generation-sequencing-analysis
#16
Maxime Pichon, Alexandre Gaymard, Laurence Josset, Martine Valette, Gilles Millat, Bruno Lina, Vanessa Escuret
INTRODUCTION: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV. METHODS: RT-qPCR was performed using the ABI7500 platform...
August 3, 2017: Antiviral Research
https://www.readbyqxmd.com/read/28771147/comparison-of-droplet-digital-pcr-and-quantitative-pcr-for-the-detection-of-salmonella-and-its-application-for-river-sediments
#17
Gulshan Singh, Ayanda Sithebe, Abimbola M Enitan, Sheena Kumari, Faizal Bux, Thor Axel Stenström
Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves...
August 2017: Journal of Water and Health
https://www.readbyqxmd.com/read/28754961/the-maintenance-of-telomere-length-in-cd28-t-cells-during-t-lymphocyte-stimulation
#18
Ejun Elijah Huang, Enzo Tedone, Ryan O'Hara, Crystal Cornelius, Tsung-Po Lai, Andrew Ludlow, Woodring E Wright, Jerry W Shay
Telomerase activity is not readily detected in resting human T lymphocytes, however upon antigen presentation, telomerase is transiently upregulated. Presently, it is not known if telomerase activation is necessary for the proliferation of T cells or for the maintenance of telomere lengths. In this study, we found that telomerase activation is not required for the short- term proliferation of T cells and that telomeres progressively shorten in a heterogeneous population of T cells, even if telomerase is detected...
July 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28754904/robust-and-accurate-digital-measurement-for-her2-amplification-in-her2-equivocal-breast-cancer-diagnosis
#19
Yuefeng Wang, Julia Y S Tsang, Yongmei Cui, Ji Cui, Ying Lin, Songli Zhao, Patrick T W Law, Sai Yin Cheung, Enders K O Ng, Gary M K Tse, Zunfu Ke
Currently, there are no recommended alternative assays for HER2 cases deemed equivocal by immunohistochemistry and fluorescent in situ hybridization. Digital PCR (ddPCR), a highly accurate method to determine DNA copy number, could be a robust alternative for clinical HER2 diagnostics. HER2 and CEP17 copy numbers were quantified using two ddPCR platforms (QX200 and RainDrop) in 102 samples of invasive breast cancers. Compared to routine assays, ddPCR gave a sensitivity and specificity of 82.8% and 97.3% respectively, with a kappa value of 0...
July 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28748500/digital-droplet-pcr-ddpcr-for-the-detection-and-quantification-of-hpv-16-18-33-and-45%C3%A2-a-short-report
#20
Gabriella Lillsunde Larsson, Gisela Helenius
PURPOSE: Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR). METHODS: Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR...
July 26, 2017: Cellular Oncology (Dordrecht)
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