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Vijayanandraj Selvaraj, Yogita Maheshwari, Subhas Hajeri, Jianchi Chen, Thomas Greg McCollum, Raymond Yokomi
Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors...
2018: PloS One
Deepani D Fernando, Simone L Reynolds, Martha Zakrzewski, Ehtesham Mofiz, Anthony T Papenfuss, Deborah Holt, Katja Fischer
BACKGROUND: Scabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites...
May 16, 2018: Parasites & Vectors
Jessica Garcia, Julien Forestier, Eric Dusserre, Anne-Sophie Wozny, Florence Geiguer, Patrick Merle, Claire Tissot, Carole Ferraro-Peyret, Frederick S Jones, Daniel L Edelstein, Valérie Cheynet, Claire Bardel, Gaelle Vilchez, Zhenyu Xu, Pierre Paul Bringuier, Marc Barritault, Karen Brengle-Pesce, Marielle Guillet, Marion Chauvenet, Brigitte Manship, Marie Brevet, Claire Rodriguez-Lafrasse, Valérie Hervieu, Sébastien Couraud, Thomas Walter, Léa Payen
CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed...
April 20, 2018: Oncotarget
Donglai Liu, Haiwei Zhou, Dawei Shi, Shu Shen, Yabin Tian, Lin Wang, Jiatao Lou, Rong Cong, Juan Lu, Henghui Zhang, Meiru Zhao, Shida Zhu, Zhisheng Cao, Ruilin Jin, Yin Wang, Xiaoni Zhang, Guohua Yang, Youchun Wang, Chuntao Zhang
Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor ( EGFR ) gene at serial allelic frequencies...
2018: Journal of Cancer
Wanlei Gao, Ting Huang, Haojun Yuan, Jun Yang, Qinghui Jin, Chunping Jia, Guoxin Mao, Jianlong Zhao
Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization...
August 1, 2018: Talanta
Safaa T Elmahalawy, Peter Halvarsson, Moa Skarin, Johan Höglund
Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT)...
April 26, 2018: International Journal for Parasitology, Drugs and Drug Resistance
Shivam H Patel, Arman Sabbaghi, Chad C Carroll
OVERVIEW: Tendon collagen fibril degradation is commonly seen in tendons of diabetics, but the mechanisms responsible for these changes remain to be elucidated. We have demonstrated that streptozotocin (STZ)-induced diabetes increases tendon cell proliferation and collagen content. In the present study, we evaluated that impact of STZ-induced diabetes on mRNA transcripts involved with collagen fibril organization, extracellular matrix (ECM) remodeling, apoptosis, and proliferation. MATERIALS AND METHODS: Rats were divided into four groups: nondiabetic (control, n = 9), 1 week (acute, n = 8) or 10 weeks of diabetes (chronic, n = 7), and 10 weeks of diabetes with insulin (insulin, n = 8)...
May 10, 2018: Connective Tissue Research
Havell Markus, Tania Contente-Cuomo, Maria Farooq, Winnie S Liang, Mitesh J Borad, Shivan Sivakumar, Simon Gollins, Nhan L Tran, Harshil D Dhruv, Michael E Berens, Alan Bryce, Aleksandar Sekulic, Antoni Ribas, Jeffrey M Trent, Patricia M LoRusso, Muhammed Murtaza
Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly...
May 9, 2018: Scientific Reports
Robert Philibert, Meesha Dogan, Amanda Noel, Shelly Miller, Brianna Krukow, Emma Papworth, Joseph Cowley, Jeffrey D Long, Steven R H Beach, Donald W Black
The tobacco use disorders are the largest preventable cause of morbidity and mortality in the world. A substantial barrier to the development of better intervention and screening measures is the lack of clinically employable biomarkers to detect the existence and extent of tobacco consumption. In prior work, we and others have shown that array based assessment of DNA methylation status at cg05575921 is a sensitive and quantitative method for assessing cigarette consumption. Unfortunately, in general, arrays are not practical clinical tools...
2018: Frontiers in Genetics
Yejin Oh, Ik-Chan Song, Jimyung Kim, Gye Cheol Kwon, Sun Hoe Koo, Seon Young Kim
BACKGROUND: We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). METHODS: Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. RESULTS: Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations)...
May 1, 2018: Clinica Chimica Acta; International Journal of Clinical Chemistry
Nataša Mehle, David Dobnik, Maja Ravnikar, Maruša Pompe Novak
RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis...
May 3, 2018: Analytical and Bioanalytical Chemistry
Michael S Hildebrand, A Simon Harvey, Stephen Malone, John A Damiano, Hongdo Do, Zimeng Ye, Lara McQuillan, Wirginia Maixner, Renate Kalnins, Bernadette Nolan, Martin Wood, Ezgi Ozturk, Nigel C Jones, Greta Gillies, Kate Pope, Paul J Lockhart, Alexander Dobrovic, Richard J Leventer, Ingrid E Scheffer, Samuel F Berkovic
Objective: To determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge-Weber syndrome (SWS) to establish a definitive molecular diagnosis. Methods: We used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations. Results: Low levels of the GNAQ mutation were detected in the brain tissue of all 4 cases-ranging from 0...
June 2018: Neurology. Genetics
Nicolas Macagno, Frédéric Fina, Nicolas Penel, Corinne Bouvier, Isabelle Nanni, Florence Duffaud, Raquel Rouah, Bruno Lacarelle, L'houcine Ouafik, Sylvie Bonvalot, Sébastien Salas
Since desmoid tumors (DT) exhibit an unpredictable clinical course, with stabilization and/or spontaneous regression, an initial "wait-and-see" policy is the new standard of care-thus, the actual challenge is to identify early factors of progression. We present a method of detection of CTNNB1 mutations using a targeted digital droplet PCR (ddPCR) on cell-free DNA (cfDNA) extracted from blood samples of 31 DT patients. Furthermore, we analyzed the correlation between DT evolution and plasmatic concentration of total and mutated cfDNA at the time of diagnosis...
April 6, 2018: Oncotarget
Aaron Ang, Eric Ouellet, Karen C Cheung, Charles Haynes
In addition to its growing use in detecting and quantifying genes and larger genomic events, the partitioning used in digital PCR can serve as a powerful tool for high-fidelity amplification of synthetic combinatorial libraries of single-stranded DNA. Sequence-diverse libraries of this type are used as a basis for selecting tight-binding aptamers against a specific target. Here we provide a detailed description of the Hi-Fi SELEX protocol for rapid and efficient DNA aptamer selection. As part of that methodology, we describe how Hi-Fi SELEX gains advantages over other aptamer selection methods in part through the use of the massive partitioning capability of digital PCR...
2018: Methods in Molecular Biology
Andrew T Ludlow, Dawne Shelton, Woodring E Wright, Jerry W Shay
Telomerase is a cellular RNA template-dependent reverse transcriptase that adds telomere repeats to the 3' ends of chromosomes. Telomerase is expressed almost universally in tumor cells (>85%) to maintain telomere length, thus providing the ability of tumor cells to avoid senescence and to have unlimited replication ability, one of the key hallmarks of cancer. ddTRAP (droplet digital Telomere Repeat Amplification Protocol) is a two-step assay with whole cell lysates that utilizes a telomerase-mediated primer extension followed by droplet digital PCR (ddPCR) detection of extended products...
2018: Methods in Molecular Biology
Nicholas J Heredia
Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence...
2018: Methods in Molecular Biology
George Karlin-Neumann, Bin Zhang, Claudia Litterst
Gene expression studies have provided one of the most accessible windows for understanding the molecular basis of cell and tissue phenotypes and how these change in response to stimuli. Current PCR-based and next generation sequencing methods offer great versatility in allowing the focused study of the roles of small numbers of genes or comprehensive profiling of the entire transcriptome of a sample at one time. Marrying of these approaches to various cell sorting technologies has recently enabled the profiling of expression in single cells, thereby increasing the resolution and sensitivity and strengthening the inferences from observed expression levels and changes...
2018: Methods in Molecular Biology
Bing Sun, Yun-Ling Zheng
Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature...
2018: Methods in Molecular Biology
Ming Yu, Tai J Heinzerling, William M Grady
Droplet digital (ddPCR) is a recent advance in PCR technology that enables the precise detection and absolute quantification of nucleic acid target sequences and that has a range of applications for both research and clinical diagnostic studies. Here, we discuss the parameters important in the design and performance of ddPCR for the detection and quantification of methylated DNA. We provide explicit instructions for conducting methylation specific ddPCR (aka MethyLight ddPCR). We also present an example that demonstrates the sensitivity and precision of the method for detecting methylated DNA in the promoter region of mir342/EVL, a potential DNA methylation biomarker for colon cancer risk...
2018: Methods in Molecular Biology
Yuichiro Miyaoka, Steven J Mayerl, Amanda H Chan, Bruce R Conklin
Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel...
2018: Methods in Molecular Biology
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