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https://www.readbyqxmd.com/read/28629339/droplet-digital-pcr-for-detection-and-quantification-of-circulating-tumor-dna-in-plasma-of-head-and-neck-cancer-patients
#1
Joost H van Ginkel, Manon M H Huibers, Robert J J van Es, Remco de Bree, Stefan M Willems
BACKGROUND: During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). METHODS: TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC)...
June 19, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28626778/single-cell-based-vector-tracing-in-patients-with-ada-scid-treated-with-stem-cell-gene-therapy
#2
Yuka Igarashi, Toru Uchiyama, Tomoko Minegishi, Sirirat Takahashi, Nobuyuki Watanabe, Toshinao Kawai, Masafumi Yamada, Tadashi Ariga, Masafumi Onodera
Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level...
September 15, 2017: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/28625519/multiplex-ultrasensitive-genotyping-of-patients-with-non-small-cell-lung-cancer-for-epidermal-growth-factor-receptor-egfr-mutations-by-means-of-picodroplet-digital-pcr
#3
Masaru Watanabe, Tomoya Kawaguchi, Shun-Ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Osamu Kawashima, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Katsuya Watanabe, Akihide Matsumura, Yasuhiro Koh
Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0...
June 7, 2017: EBioMedicine
https://www.readbyqxmd.com/read/28615231/single-nucleotide-polymorphism-leading-to-false-allelic-fraction-by-droplet-digital-pcr
#4
Eric S Christenson, William B Dalton, David Chu, Ian Waters, Karen Cravero, Daniel J Zabransky, Amy DeZern, Ben Ho Park
BACKGROUND: Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS: Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles...
June 14, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/28602964/droplet-digital-pcr-improved-the-egfr-mutation-diagnosis-with-pleural-fluid-samples-in-non-small-cell-lung-cancer-patients
#5
Xiaoyan Li, Yi Liu, Weiwei Shi, Huayan Xu, Haixu Hu, Zhengwei Dong, Guanshan Zhu, Yun Sun, Bing Liu, Hongjun Gao, Chuanhao Tang, Xiaoqing Liu
BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of nucleic acid. However, its application has not been reported in pleural fluid, which is an ideal sample source for epidermal growth factor receptor (EGFR) mutation analysis in non-small-cell lung cancer (NSCLC) patients. METHODS: The extracted DNA from supernatants of pleural fluid was selected from our sample bank and re-analyzed by our previously established ddPCR assay...
June 8, 2017: Clinica Chimica Acta; International Journal of Clinical Chemistry
https://www.readbyqxmd.com/read/28590119/parallel-evaluation-of-melting-temperatures-of-dnas-in-the-arrayed-droplets-through-the-fluorescence-from-dna-intercalators
#6
Yuzuru Shimazaki, Junko Tanaka, Yoshinobu Kohara, Masao Kamahori, Takeshi Sakamoto
Parallel evaluation of melting temperatures (Tm's) of DNA molecules in multiple floating droplets (20 μm in diameter) was demonstrated. The Tm values were evaluated from the melting curves which were observed through the fluorescence from the DNA intercalators. The Tm values measured in the droplets corresponded well to those measured in the bulk, indicating the validity of the measurement. The parallel evaluation of Tm's was realized by observing melting curves of DNAs in the different droplets at the same time using the "droplet guide", which guided and fixed the floating droplets to the designated points in the observing plane...
June 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28577941/quantitative-cell-free-circulating-egfr-mutation-concentration-is-correlated-with-tumor-burden-in-advanced-nsclc-patients
#7
Yan-Juan Zhu, Hai-Bo Zhang, Yi-Hong Liu, Fu-Li Zhang, Ya-Zhen Zhu, Yong Li, Jian-Ping Bai, Li-Rong Liu, Yan-Chun Qu, Xin Qu, Xian Chen, Yan Li, Guang-Juan Zheng
OBJECTIVES: Droplet digital polymerase chain reaction (ddPCR) has shown sufficient concordance in detecting plasma epidermal growth factor receptor (EGFR) status in non-small cell lung cancer (NSCLC), compared to tumor tissues. However, the clinical significance of the quantitative plasma mutated EGFR concentration remains unknown. The purpose of this study was to explore the relationship of plasma mutated EGFR concentration with tumor burden in advanced NSCLC patients. MATERIALS AND METHODS: Using ddPCR, plasma DNA samples prior to administration of therapies from 113 consecutive NSCLC patients were analyzed for EGFR L858R substitution and deletion of exon19 (ex19del)...
July 2017: Lung Cancer: Journal of the International Association for the Study of Lung Cancer
https://www.readbyqxmd.com/read/28562660/a-droplet-digital-pcr-ddpcr-assay-to-detect-helicoverpa-armigera-lepidoptera-noctuidae-in-bulk-trap-samples
#8
Frida A Zink, Luke R Tembrock, Alicia E Timm, Roxanne E Farris, Omaththage P Perera, Todd M Gilligan
Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H...
2017: PloS One
https://www.readbyqxmd.com/read/28552765/comprehensive-analysis-of-the-discordance-of-egfr-mutation-status-between-tumor-tissues-and-matched-circulating-tumor-dna-in-advanced-non-small-cell-lung-cancer
#9
Rui Wan, Zhijie Wang, J Jack Lee, Shuhang Wang, Qingqing Li, Fuchou Tang, Jin Wang, Yu Sun, Hua Bai, Di Wang, Jun Zhao, Jianchun Duan, Minglei Zhuo, Tongtong An, Meina Wu, Zhaoli Chen, Zhenlin Yang, Jie Wang
INTRODUCTION: This study aimed to address the underlying reasons and clinical significance of the discordant epidermal growth factor receptor mutation (EGFRm) status between tumor tissues (T) and circulating tumor DNA (ctDNA, C). METHODS: Three groups of EGFR tyrosine kinase inhibitors (EGFR-TKIs) treated patients whose EGFRm were determined by Amplification Refractory Mutation System (ARMS) were included (Group A: EGFRm T+/C+; Group B: EGFRm T-/C+; and Group C: EGFRm T+/C-)...
May 25, 2017: Journal of Thoracic Oncology
https://www.readbyqxmd.com/read/28546538/droplet-digital-pcr-versus-qpcr-for-gene-expression-analysis-with-low-abundant-targets-from-variable-nonsense-to-publication-quality-data
#10
Sean C Taylor, Genevieve Laperriere, Hugo Germain
Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower...
May 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28540249/liquid-biopsies-for-bladder-cancer
#11
EDITORIAL
Douglas G Ward, Richard T Bryan
The development of accurate urinary biomarkers for the non-invasive detection of urothelial bladder cancer (UBC) could transform patient pathways by reducing reliance on cystoscopy, and the identification of highly prognostic (or even predictive) biomarkers could better guide patient management. A number of approaches are being utilised to address these challenges in both urinary- and plasma-borne tumour DNA (tDNA), so-called "liquid biopsies". Next generation sequencing (NGS) and droplet digital PCR (ddPCR) allow detection of very low levels of such tDNA amongst a large excess of non-tumour DNA, the former permitting large mutation panels to be assessed and the latter potentially identifying ultrarare mutant alleles yet restricted for multiplexing...
April 2017: Translational Andrology and Urology
https://www.readbyqxmd.com/read/28529628/cross-platform-comparison-of-four-leading-technologies-for-detecting-egfr-mutations-in-circulating-tumor-dna-from-non-small-cell-lung-carcinoma-patient-plasma
#12
Ting Xu, Xiaozheng Kang, Xiaofang You, Liang Dai, Dequan Tian, Wanpu Yan, Yongbo Yang, Hongchao Xiong, Zhen Liang, Grace Q Zhao, Shengrong Lin, Ke-Neng Chen, Guobing Xu
Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. Here, we have compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. Two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction (ddPCR) and a next-generation sequencing (Firefly NGS) platform were included in the comparison...
2017: Theranostics
https://www.readbyqxmd.com/read/28522907/droplet-digital-pcr-for-quantification-of-itga6-in-a-stool-mrna-assay-for-the-detection-of-colorectal-cancers
#13
Elizabeth Herring, Shigeru Kanaoka, Éric Tremblay, Jean-François Beaulieu
AIM: To investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening. METHODS: ddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods...
April 28, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28497422/utility-of-urinary-circulating-tumor-dna-for-egfr-mutation-detection-in-different-stages-of-non-small-cell-lung-cancer-patients
#14
Fajiu Li, Jie Huang, Dongyuan Ji, Qinghua Meng, Chuanhai Wang, Shi Chen, Xiaojiang Wang, Zhiyang Zhu, Cheng Jiang, Yi Shi, Shuang Liu, Chenghong Li
PURPOSE: Non-invasive methods of molecular profiling for non-small cell lung cancer (NSCLC) are useful for monitoring disease progression. The aim of the current study was to ascertain if transrenal DNA is sensitive for clinical correlation and EGFR detection in NSCLC patients. METHODS: 160 patients at various stages of the disease participated and samples were collected prospectively at 2-month intervals. A baseline sample was taken before treatment commencement...
May 11, 2017: Clinical & Translational Oncology
https://www.readbyqxmd.com/read/28490568/noninvasive-detection-of-f8-int22h-related-inversions-and-sequence-variants-in-maternal-plasma-of-hemophilia-carriers
#15
Irena Hudecova, Peiyong Jiang, Joanna Davies, Y M Dennis Lo, Rezan A Kadir, Rossa W K Chiu
Direct detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among hemophilia patients, namely those involving int22h-related inversions disrupting the F8 gene, poses additional challenges due to its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia...
May 10, 2017: Blood
https://www.readbyqxmd.com/read/28487054/reduced-cost-chlamydia-trachomatis-specific-multiplex-real-time-pcr-diagnostic-assay-evaluated-for-ocular-swabs-and-use-by-trachoma-research-programmes
#16
Robert Butcher, Jo Houghton, Tamsyn Derrick, Athumani Ramadhani, Beatriz Herrera, Anna R Last, Patrick A Massae, Matthew J Burton, Martin J Holland, Chrissy H Roberts
INTRODUCTION: Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers...
May 6, 2017: Journal of Microbiological Methods
https://www.readbyqxmd.com/read/28484715/plasma-circulating-tumor-dna-levels-for-the-monitoring-of-melanoma-patients-landscape-of-available-technologies-and-clinical-applications
#17
REVIEW
Benoit Busser, Julien Lupo, Lucie Sancey, Stéphane Mouret, Patrice Faure, Joel Plumas, Laurence Chaperot, Marie Thérèse Leccia, Jean Luc Coll, Amandine Hurbin, Pierre Hainaut, Julie Charles
Melanoma is a cutaneous cancer with an increasing worldwide prevalence and high mortality due to unresectable or metastatic stages. Mutations in BRAF, NRAS, or KIT are present in more than 60% of melanoma cases, but a useful blood-based biomarker for the clinical monitoring of melanoma patients is still lacking. Thus, the analysis of circulating tumor cells (CTCs) and/or cell-free circulating tumor DNA (ctDNA) analysis from blood (liquid biopsies) appears to be a promising noninvasive, repeatable, and systemic sampling tool for detecting and monitoring melanoma...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28477149/current-and-emerging-applications-of-droplet-digital-pcr-in-oncology
#18
REVIEW
Susana Olmedillas-López, Mariano García-Arranz, Damián García-Olmo
The clinical management of cancer has evolved in recent years towards more personalized strategies that require a comprehensive knowledge of the complex molecular features involved in tumor growth and evolution, and the development of drug resistance mechanisms leading to disease progression. Droplet digital PCR (ddPCR) has become one of the most accurate and reliable tools for the examination of genetic alterations in a wide variety of cancers because of its high sensitivity and specificity. ddPCR is currently being applied for absolute allele quantification, rare mutation detection, analysis of copy number variations, DNA methylation, and gene rearrangements in different kinds of clinical samples...
May 5, 2017: Molecular Diagnosis & Therapy
https://www.readbyqxmd.com/read/28475662/twoddpcr-an-r-bioconductor-package-and-shiny-app-for-droplet-digital-pcr-analysis
#19
Anthony Chiu, Mahmood Ayub, Caroline Dive, Ged Brady, Crispin J Miller
Summary: Droplet Digital PCR (ddPCR) is a sensitive platform used to quantify specific nucleic acid molecules amplified by polymerase chain reactions. Its sensitivity makes it particularly useful for the detection of rare mutant molecules, such as those present in a sample of circulating free tumour DNA obtained from cancer patients. ddPCR works by partitioning a sample into individual droplets for which the majority contain only zero or one target molecule. Each droplet then becomes a reaction chamber for PCR, which through the use of fluorochrome labelled probes allows the target molecules to be detected by measuring the fluorescence intensity of each droplet...
May 5, 2017: Bioinformatics
https://www.readbyqxmd.com/read/28472366/androgen-receptor-gene-status-in-plasma-dna-associates-with-worse-outcome-on-enzalutamide-or-abiraterone-for-castration-resistant-prostate-cancer-a-multi-institution-correlative-biomarker-study
#20
V Conteduca, D Wetterskog, M T A Sharabiani, E Grande, M P Fernandez-Perez, A Jayaram, S Salvi, D Castellano, A Romanel, C Lolli, V Casadio, G Gurioli, D Amadori, A Font, S Vazquez-Estevez, A González Del Alba, B Mellado, O Fernandez-Calvo, M J Méndez-Vidal, M A Climent, I Duran, E Gallardo, A Rodriguez, C Santander, M I Sáez, J Puente, D Gasi Tandefelt, A Wingate, D Dearnaley, F Demichelis, U De Giorgi, E Gonzalez-Billalabeitia, G Attard
Background: There is an urgent need to identify biomarkers to guide personalized therapy in castration-resistant prostate cancer (CRPC). We aimed to clinically qualify androgen receptor (AR) gene status measurement in plasma DNA using multiplex droplet digital PCR (ddPCR) in pre- and post-chemotherapy CRPC. Methods: We optimized ddPCR assays for AR copy number and mutations and retrospectively analyzed plasma DNA from patients recruited to one of the three biomarker protocols with prospectively collected clinical data...
May 3, 2017: Annals of Oncology: Official Journal of the European Society for Medical Oncology
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