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https://www.readbyqxmd.com/read/28319152/a-digital-pcr-method-for-identifying-and-quantifying-adulteration-of-meat-species-in-raw-and-processed-food
#1
Junan Ren, Tingting Deng, Wensheng Huang, Ying Chen, Yiqiang Ge
Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination...
2017: PloS One
https://www.readbyqxmd.com/read/28303131/multiplexed-single-intact-cell-droplet-digital-pcr-music-ddpcr-method-for-specific-detection-of-enterohemorrhagic-e-coli-ehec-in-food-enrichment-cultures
#2
Tanis C McMahon, Burton W Blais, Alex Wong, Catherine D Carrillo
Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28301519/lignin-degrading-peroxidases-in-white-rot-fungus-trametes-hirsuta-072-absolute-expression-quantification-of-full-multigene-family
#3
Daria V Vasina, Konstantin V Moiseenko, Tatiana V Fedorova, Tatiana V Tyazhelova
Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader...
2017: PloS One
https://www.readbyqxmd.com/read/28274280/a-novel-duplex-ddpcr-assay-for-the-diagnosis-of-schistosomiasis-japonica-proof-of-concept-in-an-experimental-mouse-model
#4
Kosala G Weerakoon, Catherine A Gordon, Pengfei Cai, Geoffrey N Gobert, Mary Duke, Gail M Williams, Donald P McManus
The current World Health Organization strategic plan targets the elimination of schistosomiasis as a public health problem by 2025 and accurate diagnostics will play a pivotal role in achieving this goal. DNA-based detection methods provide a viable alternative to some of the commonly used tests, notably microscopy and serology, for the diagnosis of schistosomiasis. The detection of parasite cell-free DNA in different clinical samples is a recent valuable advance, which provides significant benefits for accurate disease diagnosis...
March 9, 2017: Parasitology
https://www.readbyqxmd.com/read/28269795/smn-blood-levels-in-a-porcine-model-of-spinal-muscular-atrophy
#5
Xueqian Wang, Samantha R Renusch, Sandra I Duque, Allison M Wehr, Xiaokui-Molly Mo, Vicki L McGovern, W David Arnold, Arthur H M Burghes, Stephen J Kolb
BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal recessive motor neuron disease that results in loss of spinal motor neurons, muscular weakness and, in severe cases, respiratory failure and death. SMA is caused by a deletion or mutation of the SMN1 gene and retention of the SMN2 gene that leads to low SMN expression levels.The measurement of SMN mRNA levels in peripheral blood samples has been used in SMA clinical studies as a pharmacodynamic biomarker for response to therapies designed to increase SMN levels...
2017: Journal of Neuromuscular Diseases
https://www.readbyqxmd.com/read/28268092/droplet-digital-pcr-is-a-reliable-tool-for-monitoring-minimal-residual-disease-in-acute-promyelocytic-leukemia
#6
Claudia Brunetti, Luisa Anelli, Antonella Zagaria, Angela Minervini, Crescenzio F Minervini, Paola Casieri, Nicoletta Coccaro, Cosimo Cumbo, Giuseppina Tota, Luciana Impera, Paola Orsini, Giorgina Specchia, Francesco Albano
Nested PCR (nPCR) and real-time quantitative PCR (qPCR) are well-established methods for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL). Despite their remarkable sensitivity and specificity, both methods have inherent limitations, such as qualitative MRD evaluation and relative quantification. Herein, we used droplet digital PCR (ddPCR) to monitor MRD in 21 APL patients and compared its performance with nPCR and qPCR. After assessing the limit of detection (LOD) for each technique on serial dilutions of PML-RARA bcr1 and bcr3 transcripts, a total of 48 follow-up samples were analyzed and the results compared...
March 4, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28267558/rapid-detection-of-donor-cell-free-dna-in-lung-transplant-recipients-with-rejections-using-donor-recipient-hla-mismatch
#7
Jun Zou, Brian Duffy, Michael Slade, Andrew Lee Young, Nancy Steward, Ramsey Hachem, T Mohanakumar
Fiberoptic bronchoscopy and transbronchial lung biopsy are currently the gold standard for detection of acute rejection following human lung transplantation (LTx). However, these surveillance procedures are expensive and invasive. Up to now, there are few new methods that have demonstrated clinical utility for detecting early stages of rejection following human lung transplantation. We optimized and technically validated a novel method to quantify donor-derived circulating cell free DNA (DcfDNA) that can be used as an early biomarker for lung allograft rejection...
March 4, 2017: Human Immunology
https://www.readbyqxmd.com/read/28259593/using-an-endoscopic-distal-cap-to-collect-pancreatic-fluid-from-the-ampulla-with-video
#8
Masaya Suenaga, Yoshihiko Sadakari, Jose Alejandro Almario, Michael Borges, Anne-Marie Lennon, Eun-Ji Shin, Marcia Irene Canto, Michael Goggins
BACKGROUND AND AIMS: Duodenal collections of pancreatic fluid can be used as a source of mutations and other markers of pancreatic ductal neoplasia, but admixing pancreatic juice with duodenal contents lowers the concentrations of mutations. Collecting pancreatic fluid directly from the ampulla could yield a purer sample of pancreatic fluid. METHODS: We used an endoscopic distal cap attachment to "cap" the ampulla and collect secretin-stimulated pancreatic fluid samples for 5 minutes from 81 patients undergoing pancreatic evaluation as part of the Cancer of the Pancreas Screening studies...
March 1, 2017: Gastrointestinal Endoscopy
https://www.readbyqxmd.com/read/28205231/analysis-of-ctdna-to-predict-prognosis-and-monitor-treatment-responses-in-metastatic-pancreatic-cancer-patients
#9
He Cheng, Chen Liu, Jiahao Jiang, Guopei Luo, Yu Lu, Kaizhou Jin, Meng Guo, Zhenzhen Zhang, Jin Xu, Liang Liu, Quanxing Ni, Xianjun Yu
Cell-free circulating tumor DNA (ctDNA) in plasma has been used as a potential noninvasive biomarker for various tumors. Our study was performed to evaluate the clinical implications of ctDNA detection in patients with metastatic pancreatic cancer. First, we attempted to prospectively screen a panel of 60 genes in cell-free DNA (cfDNA) from ten metastatic pancreatic cancer patients via exome sequencing. Second, droplet digital PCR (ddPCR) was used to identify potential mutations in a cohort of 188 patients with metastatic pancreatic cancer...
February 16, 2017: International Journal of Cancer. Journal International du Cancer
https://www.readbyqxmd.com/read/28190454/somatic-map2k1-mutations-are-associated-with-extracranial-arteriovenous-malformation
#10
Javier A Couto, August Y Huang, Dennis J Konczyk, Jeremy A Goss, Steven J Fishman, John B Mulliken, Matthew L Warman, Arin K Greene
Arteriovenous malformation (AVM) is a fast-flow, congenital vascular anomaly that may arise anywhere in the body. AVMs typically progress, causing destruction of surrounding tissue and, sometimes, cardiac overload. AVMs are difficult to control; they often re-expand after embolization or resection, and pharmacologic therapy is unavailable. We studied extracranial AVMs in order to identify their biological basis. We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) on AVM tissue from affected individuals...
March 2, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28183140/correlation-between-pik3ca-mutations-in-cell-free-dna-and-everolimus-efficacy-in-hr-her2-advanced-breast-cancer-results-from-bolero-2
#11
Mary Ellen Moynahan, David Chen, Wei He, Patricia Sung, Aliaksandra Samoila, Daoqi You, Trusha Bhatt, Parul Patel, Francois Ringeisen, Gabriel N Hortobagyi, Jose Baselga, Sarat Chandarlapaty
BACKGROUND: The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation. METHODS: PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm. RESULTS: Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0...
March 14, 2017: British Journal of Cancer
https://www.readbyqxmd.com/read/28154880/accurate-quantitation-of-circulating-cell-free-mitochondrial-dna-in-plasma-by-droplet-digital-pcr
#12
Wei Ye, Xiaojun Tang, Chu Liu, Chaowei Wen, Wei Li, Jianxin Lyu
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ(0)) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA...
February 2, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28153953/evaluation-of-a-fast-and-fully-automated-platform-to-diagnose-egfr-and-kras-mutations-in-formalin-fixed-and-paraffin-embedded-non-small-cell-lung-cancer-samples-in-less-than-one-day
#13
Laetitia Lambros, Charline Caumont, Briac Guibourg, Fanny Barel, Isabelle Quintin-Roué, Pascale Marcorelles, Jean-Philippe Merlio, Arnaud Uguen
AIMS: Searching for EGFR and KRAS mutations within non-small cell lung carcinoma (NSCLC) samples remains time-consuming and can delay treatment choices in patients with acute deterioration. We evaluated the performances of the fully automated Idylla platform to quickly detect these mutations in NSCLC samples. METHODS: We used the Idylla EGFR Mutation Assay and the Idylla KRAS Mutation Test to analyse 18 formalin-fixed paraffin-embedded NSCLC tumour samples with known EGFR and KRAS mutation status according to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) for EGFRT790M mutations...
February 2, 2017: Journal of Clinical Pathology
https://www.readbyqxmd.com/read/28152506/detection-fidelity-of-ar-mutations-in-plasma-derived-cell-free-dna
#14
Alexa Goldstein, Patricia Valda Toro, Justin Lee, John L Silberstein, Mary Nakazawa, Ian Waters, Karen Cravero, David Chu, Rory L Cochran, Minsoo Kim, Daniel Shinn, Samantha Torquato, Robert M Hughes, Aparna Pallavajjala, Michael A Carducci, Channing J Paller, Samuel R Denmeade, Bruce Kressel, Bruce J Trock, Mario A Eisenberger, Emmanuel S Antonarakis, Ben H Park, Paula J Hurley
Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%...
January 31, 2017: Oncotarget
https://www.readbyqxmd.com/read/28135250/cell-to-cell-heterogeneity-of-ewsr1-fli1-activity-determines-proliferation-migration-choices-in-ewing-sarcoma-cells
#15
G-A Franzetti, K Laud-Duval, W van der Ent, A Brisac, M Irondelle, S Aubert, U Dirksen, C Bouvier, G de Pinieux, E Snaar-Jagalska, P Chavrier, O Delattre
Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1(high) states, characterized by highly active cell proliferation, and EWSR1-FLI1(low) states where cells have a strong propensity to migrate, invade and metastasize...
January 30, 2017: Oncogene
https://www.readbyqxmd.com/read/28125683/differences-in-amy1-gene-copy-numbers-derived-from-blood-buccal-cells-and-saliva-using-quantitative-and-droplet-digital-pcr-methods-flagging-the-pitfall
#16
Delicia Shu Qin Ooi, Verena Ming Hui Tan, Siong Gim Ong, Yiong Huak Chan, Chew Kiat Heng, Yung Seng Lee
INTRODUCTION: The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. METHODS: Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant...
2017: PloS One
https://www.readbyqxmd.com/read/28120216/a-somatic-gna11-mutation-is-associated-with-extremity-capillary-malformation-and-overgrowth
#17
Javier A Couto, Ugur M Ayturk, Dennis J Konczyk, Jeremy A Goss, August Y Huang, Steve Hann, Jennifer L Reeve, Marilyn G Liang, Joyce Bischoff, Matthew L Warman, Arin K Greene
BACKGROUND: Capillary malformation is a cutaneous vascular anomaly that is present at birth, darkens over time, and can cause overgrowth of tissues beneath the stain. The lesion is caused by a somatic activating mutation in GNAQ. In a previous study, we were unable to identify a GNAQ mutation in patients with a capillary malformation involving an overgrown lower extremity. We hypothesized that mutations in GNA11 or GNA14, genes closely related to GNAQ, also may cause capillary malformations...
January 24, 2017: Angiogenesis
https://www.readbyqxmd.com/read/28114422/a-comparative-study-of-serum-exosome-isolation-using-differential-ultracentrifugation-and-three-commercial-reagents
#18
Inas Helwa, Jingwen Cai, Michelle D Drewry, Arthur Zimmerman, Michael B Dinkins, Mariam Lotfy Khaled, Mutsa Seremwe, W Michael Dismuke, Erhard Bieberich, W Daniel Stamer, Mark W Hamrick, Yutao Liu
Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors...
2017: PloS One
https://www.readbyqxmd.com/read/28104621/high-prevalence-of-mutant-kras-in-circulating-exosome-derived-dna-from-early-stage-pancreatic-cancer-patients
#19
K Allenson, J Castillo, F A San Lucas, G Scelo, D U Kim, V Bernard, G Davis, T Kumar, M Katz, M J Overman, L Foretova, E Fabianova, I Holcatova, V Janout, F Meric-Bernstam, P Gascoyne, I Wistuba, G Varadhachary, P Brennan, S Hanash, D Li, A Maitra, H Alvarez
BACKGROUND: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR (ddPCR) was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13...
January 18, 2017: Annals of Oncology: Official Journal of the European Society for Medical Oncology
https://www.readbyqxmd.com/read/28100495/probe-free-digital-pcr-quantitative-methodology-to-measure-donor-specific-cell-free-dna-after-solid-organ-transplantation
#20
Su Kah Goh, Vijayaragavan Muralidharan, Christopher Christophi, Hongdo Do, Alexander Dobrovic
BACKGROUND: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer to Hybridize across an Allelic BREakpoint-PCR)...
January 18, 2017: Clinical Chemistry
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