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Digital PCR

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https://www.readbyqxmd.com/read/29141311/-study-on-mosaicism-of-scn1a-gene-mutation-in-parents-of-children-with-dravet-syndrome
#1
A J Liu, X X Yang, X J Xu, Q X Wu, X J Tian, X L Yang, X R Wu, L P Wei, Y H Zhang
Objective: To investigate the clinical phenotypes and the mutant allele proportion of parents with SCN1A gene mutation mosaicism of Dravet syndrome (DS) children, thus to provide guidance for family reproduction and prenatal diagnosis. Method: The clinical data and peripheral blood DNA samples of DS patients with a SCN1A gene mutation proved by Sanger sequencing were collected prospectively from February 2005 to November 2016 in Department of Pediatrics, Peking University First Hospital. The same mutation was searched in parents and other available relatives...
November 2, 2017: Zhonghua Er Ke za Zhi. Chinese Journal of Pediatrics
https://www.readbyqxmd.com/read/29138500/circulating-androgen-receptor-combined-with-18f-fluorocholine-pet-ct-metabolic-activity-and-outcome-to-androgen-receptor-signalling-directed-therapies-in-castration-resistant-prostate-cancer
#2
V Conteduca, E Scarpi, P Caroli, S Salvi, C Lolli, S L Burgio, C Menna, G Schepisi, S Testoni, G Gurioli, G Paganelli, V Casadio, F Matteucci, U De Giorgi
The association between choline uptake and androgen receptor (AR) expression is suggested by the upregulation of choline kinase-alpha in prostate cancer. Recently, detection of AR aberration in cell-free DNA as well as early 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) were associated with outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone and enzalutamide. We aimed to make a direct comparison between circulating AR copy number (CN) and choline uptake at FCH-PET/CT...
November 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29136689/-detection-of-epidermal-growth-factor-receptor-gene-mutations-in-different-types-of-non-small-cell-lung-cancer-by-droplet-digital-pcr-and-amplification-refractory-mutation-system
#3
R Li, S B Ye, Y He, X Wang, N Wu, Q Y Xia, Q Shen, S S Shi
Objective: To compare amplification refractory mutation system(ARMS) and droplet digital PCR (ddPCR) in the detection of epidermal growth factor receptor (EGFR) gene mutations in patients with non-small cell lung cancer (NSCLC), and to investigate the clinical value of ddPCR. Methods: A total of 79 specimens of NSCLC, including 22 cases of cell block, 18 cases of surgical specimens, 12 cases of biopsy specimens and 27 cases of plasma samples, were analyzed for the mutation status of EGFR gene by ARMS and droplet digital PCR method...
November 8, 2017: Zhonghua Bing Li Xue za Zhi Chinese Journal of Pathology
https://www.readbyqxmd.com/read/29130947/application-of-digital-pcr-in-detecting-human-diseases-associated-gene-mutation
#4
Yu Tong, Shizhen Shen, Hui Jiang, Zhi Chen
Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research...
October 18, 2017: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/29122835/multiple-hotspot-mutations-scanning-by-single-droplet-digital-pcr
#5
Charles Decraene, Amanda Silveira, François-Clément Bidard, Audrey Vallee, Marc Michel, Samia Melaabi, Anne Vincent-Salomon, Adrien Saliou, Alexandre Houy, Maud Milder, Olivier Lantz, Marc Ychou, Marc G Denis, Jean-Yves Pierga, Marc-Henri Stern, Charlotte Proudhon
BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR)(12), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS() exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes...
November 9, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/29118176/highly-sensitive-detection-of-isoniazid-heteroresistance-in-mycobacterium-tuberculosis-by-deepmelt-assay
#6
Bin Liang, Yaoju Tan, Zi Li, Xueshan Tian, Chen Du, Hui Li, Guoli Li, Xiangyang Yao, Zhongan Wang, Ye Xu, Qingge Li
Detection of heteroresistance of Mycobacterium tuberculosis (MTB) remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less abundant mutants through selective clamping of the wild-type in mixed populations. Singleplex DeepMelt assay detected 0.01% katG S315T in 10(5) MTB genomes/μL. Multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 10(4) MTB genomes/μL...
November 8, 2017: Journal of Clinical Microbiology
https://www.readbyqxmd.com/read/29116112/a-comparison-of-mrna-sequencing-with-random-primed-and-3-directed-libraries
#7
Yuguang Xiong, Magali Soumillon, Jie Wu, Jens Hansen, Bin Hu, Johan G C van Hasselt, Gomathi Jayaraman, Ryan Lim, Mehdi Bouhaddou, Loren Ornelas, Jim Bochicchio, Lindsay Lenaeus, Jennifer Stocksdale, Jaehee Shim, Emilda Gomez, Dhruv Sareen, Clive Svendsen, Leslie M Thompson, Milind Mahajan, Ravi Iyengar, Eric A Sobie, Evren U Azeloglu, Marc R Birtwistle
Creating a cDNA library for deep mRNA sequencing (mRNAseq) is generally done by random priming, creating multiple sequencing fragments along each transcript. A 3'-end-focused library approach cannot detect differential splicing, but has potentially higher throughput at a lower cost, along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) that correct PCR bias. Here, we compare an implementation of such a 3'-digital gene expression (3'-DGE) approach with "conventional" random primed mRNAseq...
November 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29115895/droplet-based-digital-pcr-and-next-generation-sequencing-for-monitoring-circulating-tumor-dna-a-cancer-diagnostic-perspective
#8
Mathilde Postel, Alice Roosen, Pierre Laurent-Puig, Valerie Taly, Shu-Fang Wang-Renault
Early detection of cancers through the analysis of ctDNA could have a significant impact on morbidity and mortality of cancer patients. However, using ctDNA for early cancer diagnosis is challenging partly due to the low amount of tumor DNA released in the circulation and its dilution within DNA originating from non-tumor cells. Development of new technologies such as droplet-based digital PCR (ddPCR) or optimized next generation sequencing (NGS) has greatly improved the sensitivity, specificity and precision for the detection of rare sequences...
November 8, 2017: Expert Review of Molecular Diagnostics
https://www.readbyqxmd.com/read/29110585/cell-free-dna-levels-and-correlation-to-stage-and-outcome-following-treatment-of-locally-advanced-rectal-cancer
#9
Anders Kindberg Boysen, Yvonne Wettergren, Boe Sandahl Sorensen, Helena Taflin, Bengt Gustavson, Karen-Lise Garm Spindler
Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery...
November 2017: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
https://www.readbyqxmd.com/read/29110175/optimization-and-verification-of-droplet-digital-pcr-even-specific-methods-for-the-quantification-of-gm-maize-das1507-and-nk603
#10
Katarzyna Grelewska-Nowotko, Magdalena Żurawska-Zajfert, Ewelina Żmijewska, Sławomir Sowa
In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform...
November 6, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/29108273/sensitive-droplet-digital-pcr-method-for-detection-of-tert-promoter-mutations-in-cell-free-dna-from-patients-with-metastatic-melanoma
#11
Ashleigh C McEvoy, Leslie Calapre, Michelle R Pereira, Tindaro Giardina, Cleo Robinson, Muhammad A Khattak, Tarek M Meniawy, Antonia L Pritchard, Nicholas K Hayward, Benhur Amanuel, Michael Millward, Melanie Ziman, Elin S Gray
Background: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis. Methods: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations...
October 3, 2017: Oncotarget
https://www.readbyqxmd.com/read/29107003/elevated-wall-tension-initiates-interleukin-6-expression-and-abdominal-aortic-dilation
#12
Adam W Akerman, Robert E Stroud, Ryan W Barrs, R Tyler Grespin, Lindsay T McDonald, R Amanda C LaRue, Rupak Mukherjee, John S Ikonomidis, Jeffery A Jones, Jean Marie Ruddy
INTRODUCTION: Hypertension (HTN) has long been associated with abdominal aortic aneurysm (AAA) development and these cardiovascular pathologies are biochemically characterized by elevated plasma levels of Angiotensin-II (AngII) as well as interleukin-6 (IL-6). A biologic relationship between HTN and AAA has not been established, however. Accordingly, the objective of this study was to evaluate whether elevated tension may initiate IL-6 production to accumulate monocyte/macrophages and promote dilation of the abdominal aorta (AA)...
October 26, 2017: Annals of Vascular Surgery
https://www.readbyqxmd.com/read/29105393/isolation-characterization-and-osteogenic-potential-of-mouse-digit-tip-blastema-cells-in-comparison-with-bone-marrow-derived-mesenchymal-stem-cells-in-vitro
#13
Leila Taghiyar, Samaneh Hosseini, Mahdi Hesaraki, Forough Azam Sayahpour, Nasser Aghdami, Mohamadreza Baghaban Eslaminejad
OBJECTIVES: Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages...
January 2018: Cell Journal
https://www.readbyqxmd.com/read/29098316/plasma-circular-rna-profiling-of-patients-with-gastric-cancer-and-their-droplet-digital-rt-pcr-detection
#14
Tianwen Li, Yongfu Shao, Liyun Fu, Yi Xie, Linwen Zhu, Weiliang Sun, Rui Yu, Bingxiu Xiao, Junming Guo
To observe the diagnostic values of circular RNAs (circRNAs), their expression profiles between gastric cancer patients' plasma and healthy controls were first assessed by circRNA microarray. Then, circRNA levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR), respectively. A total of 343 differentially expressed circRNAs were found. The top 10 elevated circRNAs in patients were hsa_circ_0088300, hsa_circ_0075825, hsa_circ_0019172, hsa_circ_0000220, hsa_circ_0035277, hsa_circ_0000301, hsa_circ_0000189, hsa_circ_0090080, hsa_circ_0001888, and hsa_circ_0001874...
November 2, 2017: Journal of Molecular Medicine: Official Organ of the "Gesellschaft Deutscher Naturforscher und Ärzte"
https://www.readbyqxmd.com/read/29097507/noninvasive-prenatal-diagnosis-of-single-gene-disorders-by-use-of-droplet-digital-pcr
#15
Joan Camunas-Soler, Hojae Lee, Louanne Hudgins, Susan R Hintz, Yair J Blumenfeld, Yasser Y El-Sayed, Stephen R Quake
BACKGROUND: Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice. METHODS: We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma...
November 2, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/29094381/platelets-harbor-prostate-cancer-biomarkers-and-the-ability-to-predict-therapeutic-response-to-abiraterone-in-castration-resistant-patients
#16
Lee-Ann Tjon-Kon-Fat, Marie Lundholm, Mona Schröder, Thomas Wurdinger, Camilla Thellenberg-Karlsson, Anders Widmark, Pernilla Wikström, Rolf Jonas Andreas Nilsson
BACKGROUND: Novel therapies for castration resistant prostate cancer (CRPC) have been introduced in the clinic with possibilities for individualized treatment plans. Best practice of those expensive drugs requires predictive biomarker monitoring. This study used circulating biomarker analysis to follow cancer-derived transcripts implicated in therapy resistance. METHOD: The isolated platelet population of blood samples and digital-PCR were used to identify selected biomarker transcripts in patients with CRPC prior chemo- or androgen synthesis inhibiting therapy...
November 2, 2017: Prostate
https://www.readbyqxmd.com/read/29093617/detection-of-kras-g12d-in-colorectal-cancer-stool-by-droplet-digital-pcr
#17
Susana Olmedillas-López, Dennis César Lévano-Linares, Carmen Laura Aúz Alexandre, Luz Vega-Clemente, Edurne León Sánchez, Alejandro Villagrasa, Jaime Ruíz-Tovar, Mariano García-Arranz, Damián García-Olmo
AIM: To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS: In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation...
October 21, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/29089331/prevalence-of-aflatoxin-associated-tp53r249s-mutation-in-hepatocellular-carcinoma-in-hispanics-in-south-texas
#18
Jingjing Jiao, Weibo Niu, Ying Wang, Keith A Baggerly, Yuanqing Ye, Xifeng Wu, Dewitt Davenport, Jose Luis Almeda, Monica M Betancourt-Garcia, R Armour Forse, Heather L Stevenson, Gordon P Watt, Joseph B McCormick, Susan P Fisher-Hoch, Laura Beretta
We aimed to determine whether aflatoxin dietary exposure plays a role in the high incidence of hepatocellular carcinoma (HCC) observed among Hispanics in South Texas.We measured TP53R249S somatic mutation, hallmark of aflatoxin etiology in HCC, using droplet digital PCR and restriction fragment length polymorphism. TP53R249S mutation was detected in 3 out of 41 HCC tumors from Hispanics in South Texas (7.3%). We also measured TP53R249S mutation in plasma cell free DNA (cfDNA) from 218 HCC patients and 96 Hispanic subjects with advanced fibrosis or cirrhosis, from South Texas...
October 31, 2017: Cancer Prevention Research
https://www.readbyqxmd.com/read/29083143/robust-multiplexed-clustering-and-denoising-of-digital-pcr-assays-by-data-gridding
#19
Billy T Lau, Christina Wood-Bouwens, Hanlee P Ji
Digital PCR (dPCR) relies on the analysis of individual partitions to accurately quantify nucleic acid species. The most widely used analysis method requires manual clustering through individual visual inspection. Some automated analysis methods have emerged but do not robustly account for multiplexed targets, low target concentration, and assay noise. In this study, we describe an open source analysis software called Calico that uses "data gridding" to increase the sensitivity of clustering toward small clusters...
November 7, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29071363/inter-laboratory-analysis-of-selected-genetically-modified-plant-reference-materials-with-digital-pcr
#20
David Dobnik, Tina Demšar, Ingrid Huber, Lars Gerdes, Sylvia Broeders, Nancy Roosens, Frederic Debode, Gilbert Berben, Jana Žel
Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems...
October 25, 2017: Analytical and Bioanalytical Chemistry
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