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https://www.readbyqxmd.com/read/28802157/droplet-digital-pcr-for-rapid-enumeration-of-viral-genomes-and-particles-from-cells-and-animals-infected-with-orthopoxviruses
#1
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/28801547/early-changes-in-plasma-dna-levels-of-mutant-kras-as-a-sensitive-marker-of-response-to-chemotherapy-in-pancreatic-cancer
#2
Marzia Del Re, Caterina Vivaldi, Eleonora Rofi, Enrico Vasile, Mario Miccoli, Chiara Caparello, Paolo Davide d'Arienzo, Lorenzo Fornaro, Alfredo Falcone, Romano Danesi
Pancreatic cancer (PDAC) is still lacking of reliable markers to monitor tumor response. CA 19-9 is the only biomarker approved, despite it has several limitations in sensitivity and specificity. Since mutations of KRAS occur in more than 90% of tumors, its detection in circulating free tumor DNA (cftDNA) could represent a biomarker to monitor chemotherapy response. Twenty-seven advanced PDAC patients given first-line 5-fluorouracil, irinotecan and oxaliplatin or gemcitabine and nab-paclitaxel were enrolled...
August 11, 2017: Scientific Reports
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
#3
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/28794817/rapid-chemical-free-breaking-of-microfluidic-emulsions-with-a-hand-held-antistatic-gun
#4
Mohsen Karbaschi, Payam Shahi, Adam R Abate
Droplet microfluidics can form and process millions of picoliter droplets with speed and ease, allowing the execution of huge numbers of biological reactions for high-throughput studies. However, at the conclusion of most experiments, the emulsions must be broken to recover and analyze their contents. This is usually achieved with demulsifiers, like perfluorooctanol and chloroform, which can interfere with downstream reactions and harm cells. Here, we describe a simple approach to rapidly and efficiently break microfluidic emulsions, which requires no chemicals...
July 2017: Biomicrofluidics
https://www.readbyqxmd.com/read/28790159/functional-imaging-and-circulating-biomarkers-of-response-to-regorafenib-in-treatment-refractory-metastatic-colorectal-cancer-patients-in-a-prospective-phase-ii-study
#5
Khurum Khan, Mihaela Rata, David Cunningham, Dow-Mu Koh, Nina Tunariu, Jens C Hahne, George Vlachogiannis, Somaieh Hedayat, Silvia Marchetti, Andrea Lampis, Mahnaz Darvish Damavandi, Hazel Lote, Isma Rana, Anja Williams, Suzanne A Eccles, Elisa Fontana, David Collins, Zakaria Eltahir, Sheela Rao, David Watkins, Naureen Starling, Jan Thomas, Eleftheria Kalaitzaki, Nicos Fotiadis, Ruwaida Begum, Maria Bali, Massimo Rugge, Eleanor Temple, Matteo Fassan, Ian Chau, Chiara Braconi, Nicola Valeri
OBJECTIVE: Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need for better patient selection. DESIGN: Patients with RAS mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (K(trans)), enhancing fraction (EF) and their product KEF (summarised median values of K(trans)× EF) were generated...
August 8, 2017: Gut
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
#6
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/28778025/comparison-of-esr1-mutations-in-tumor-tissue-and-matched-plasma-samples-from-metastatic-breast-cancer-patients
#7
Takashi Takeshita, Yutaka Yamamoto, Mutsuko Yamamoto-Ibusuki, Mai Tomiguchi, Aiko Sueta, Keiichi Murakami, Yoko Omoto, Hirotaka Iwase
BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples)...
July 31, 2017: Translational Oncology
https://www.readbyqxmd.com/read/28771608/detection-and-quantification-of-beef-and-pork-materials-in-meat-products-by-duplex-droplet-digital-pcr
#8
Yicun Cai, Yuping He, Rong Lv, Hongchao Chen, Qiang Wang, Liangwen Pan
Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef (Bos taurus) and pork (Sus scrofa) in a single digital PCR reaction tube...
2017: PloS One
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
#9
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/28765952/mtor-signaling-promotes-foam-cell-formation-and-inhibits-foam-cell-egress-through-suppressing-the-sirt1-signaling-pathway
#10
Haixiang Zheng, Yucai Fu, Yusheng Huang, Xinde Zheng, Wei Yu, Wei Wang
Atherosclerosis (AS) is a chronic immuno‑inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C‑C chemokine receptor type 7/nuclear factor‑κB (LXR‑CCR7/NF‑κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1‑LXR‑CCR7/NF‑κB signaling (SIRT1 signaling) in AS pathogenesis...
September 2017: Molecular Medicine 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
#11
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)
https://www.readbyqxmd.com/read/28743297/identification-of-reference-mirnas-in-plasma-useful-for-the-study-of-oestrogen-responsive-mirnas-associated-with-acquired-protein-s-deficiency-in-pregnancy
#12
J W Tay, I James, Q W Hughes, J Y Tiao, R I Baker
BACKGROUND: Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users...
July 25, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/28742791/correlation-between-circulating-tumor-dna-levels-and-response-to-tyrosine-kinase-inhibitors-tki-treatment-in-non-small-cell-lung-cancer
#13
Zhangjing Wei, Wenyue Wang, Zitan Shu, Xue Zhou, Yanfang Zhang
BACKGROUND Clinical monitoring of EGFR-positive NSCLC patients is important to gauge treatment response. The current study addresses the usage of circulating tumor DNA (ctDNA) as a prognostic marker during treatment of first-generation TKIs. MATERIAL AND METHODS Serial samplings of peripheral blood from 200 EGFR-positive NSCLC patients were taken. Baseline ctDNA quantification was conducted by digital droplet PCR before TKI treatment was administered and compared to primary biopsies. Thereafter blood sampling at different treatment cycles were measured and assessed for its prognostic and predictive value...
July 25, 2017: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
https://www.readbyqxmd.com/read/28740406/conventional-real-time-pcr-based-detection-of-t790m-using-tumor-tissue-or-blood-in-patients-with-egfr-tki-resistant-nsclc
#14
Ya-Lan Wu, Rui-Zhan Tong, Yan Zhang, Bin-Bin Hu, Ke Zheng, Zhen-Yu Ding, Feng Peng, You-Ling Gong, Yong-Mei Liu, You Lu
Blood biopsy has many advantages over tissue biopsy for diagnosing acquired T790M mutation in patients with non-small-cell lung cancer, such as being less risky and painful. New techniques with high sensitivity (eg, droplet digital PCR) show promising results during blood biopsy, but the positive rates of identification are still quite unclear. Whether there are other factors, except technology, affecting the results of blood biopsy is unclear. In this study, we used conventional amplification refractory mutation system to detect tumor tissue or blood for T790M mutation in patients clinically resistant to tyrosine kinase inhibitors...
2017: OncoTargets and Therapy
https://www.readbyqxmd.com/read/28737741/rapidly-inducible-cas9-and-dsb-ddpcr-to-probe-editing-kinetics
#15
John C Rose, Jason J Stephany, William J Valente, Bridget M Trevillian, Ha V Dang, Jason H Bielas, Dustin J Maly, Douglas M Fowler
We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs...
July 24, 2017: Nature Methods
https://www.readbyqxmd.com/read/28733559/dynamics-of-egfr-mutations-in-plasma-recapitulates-the-clinical-response-to-egfr-tkis-in-nsclc-patients
#16
Liwen Xiong, Shaohua Cui, Jingyan Ding, Yun Sun, Longfu Zhang, Yizhuo Zhao, Aiqin Gu, Tianqing Chu, Huimin Wang, Hua Zhong, Xin Ye, Yi Gu, Xin Zhang, Min Hu, Liyan Jiang
OBJECTIVES: Genomic profiling using plasma cell-free DNA (cfDNA) represents a non-invasive alternative to tumor re-biopsy, which is challenging in clinical practice. The feasibility of dynamically monitoring epidermal growth factor receptor (EGFR) mutation status using serial plasma samples from non-small cell lung cancer (NSCLC) patients treated by tyrosine kinase inhibitors (TKIs) and its application in tracking clinical response and detection of resistance were investigated. PATIENTS AND METHODS: Forty-five NSCLC patients with EGFR mutation-positive pre-TKI plasma and at least two post-TKI plasma collections were recruited to this study...
July 10, 2017: Oncotarget
https://www.readbyqxmd.com/read/28729336/prenatal-diagnosis-by-droplet-digital-pcr
#17
Margaret V Ragni
No abstract text is available yet for this article.
July 20, 2017: Blood
https://www.readbyqxmd.com/read/28729136/heparinase-enables-reliable-quantification-of-circulating-tumor-dna-from-heparinized-plasma-samples-by-droplet-digital-pcr
#18
David Sefrioui, Ludivine Beaussire, Florian Clatot, Julien Delacour, Anne Perdrix, Thierry Frebourg, Pierre Michel, Frédéric Di Fiore, Nasrin Sarafan-Vasseur
BACKGROUND: Heparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential "liquid biopsy". We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis. METHODS: Plasma samples were collected in heparinized (n=194) and EDTA (n=8) tubes from hormone receptor-positive metastatic breast cancer (HR+MBC) (n=144) and pancreatic adenocarcinoma (PA) patients (n=50)...
July 17, 2017: Clinica Chimica Acta; International Journal of Clinical Chemistry
https://www.readbyqxmd.com/read/28729070/abc-transporters-in-the-liver-fluke-opisthorchis-felineus
#19
Viatcheslav A Mordvinov, Nikita I Ershov, Darya S Pirozhkova, Yuri V Pakharukov, Maria Y Pakharukova
ATP-binding cassette transporters (ABC transporters) are essential components of normal cellular physiological machinery in all eukaryotic and prokaryotic species, including parasites. Some ABC transporters, e.g., P-glycoproteins, are involved in the efflux of toxins and xenobiotics from the cell. At present, nothing is known about ABC transporter genes in epidemiologically important liver flukes from the Opisthorchiidae family, including European liver fluke Opisthorchis felineus. Opisthorchiasis caused by O...
July 17, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28725028/detection-and-quantitation-of-copy-number-variation-in-the-voltage-gated-sodium-channel-gene-of-the-mosquito-culex-quinquefasciatus
#20
Walter Fabricio Silva Martins, Krishanthi Subramaniam, Keith Steen, Henry Mawejje, Triantafillos Liloglou, Martin James Donnelly, Craig Stephen Wilding
Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides...
July 19, 2017: Scientific Reports
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