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Jing Zhao, Xin Ye, Yan Xu, Minjiang Chen, Wei Zhong, Yun Sun, Zhenfan Yang, Guanshan Zhu, Yi Gu, Mengzhao Wang
PURPOSE: Central nervous system (CNS) is the prevalent site for metastases in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-relapsed NSCLC patients. To understand the EGFR mutation status in paired cerebrospinal fluid (CSF) and plasma samples after EGFR-TKI treatment failure might be useful to guide the treatment of intra- and extracranial tumors in those patients. METHODS: Paired CSF and plasma samples were collected from seven NSCLC patients with CNS metastases after EGFR-TKI failure...
October 21, 2016: Cancer Chemotherapy and Pharmacology
Huan-Ting Lin, Takashi Okumura, Yukinori Yatsuda, Satoru Ito, Hiromitsu Nakauchi, Makoto Otsu
Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT...
October 2016: Human Gene Therapy Methods
Daniel C Danila, Aliaksandra Samoila, Chintan Patel, Nicole Schreiber, Amrita Herkal, Aseem Anand, Diogo Bastos, Glenn Heller, Martin Fleisher, Howard I Scher
Circulating tumor cell (CTC) number measured with the CellSearch assay is prognostic for survival in metastatic castration-resistant prostate cancer before and after therapy. Using a standard operating protocol for sample collection, processing, and analysis, we compared detection rates of CellSearch performed using US Food and Drug Administration-cleared methodology with a second positive selection assay, AdnaTest, and a nonselection polymerase chain reaction (PCR)-based (direct detection PCR [DDPCR]) assay in 55 blood samples from 47 men with progressive metastatic castration-resistant prostate cancer...
September 2016: Cancer Journal
Marzia Del Re, Elisa Biasco, Stefania Crucitta, Lisa Derosa, Eleonora Rofi, Cinzia Orlandini, Mario Miccoli, Luca Galli, Alfredo Falcone, Guido W Jenster, Ron H van Schaik, Romano Danesi
BACKGROUND: The androgen receptor splice variant 7 (AR-V7) is associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). Due to limitations of the methods available for AR-V7 analysis, the identification of a reliable detection method may facilitate the use of this biomarker in clinical practice. OBJECTIVE: To confirm AR-V7 as a predictor of resistance to hormonal therapy and develop a new approach to assess AR-V7 by highly sensitive digital droplet polymerase chain reaction (ddPCR) in plasma-derived exosomal RNA...
August 26, 2016: European Urology
Dean Attali, Roza Bidshahri, Charles Haynes, Jennifer Bryan
Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr - a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface...
2016: F1000Research
Takahiro Kishikawa, Motoyuki Otsuka, Takeshi Yoshikawa, Motoko Ohno, Keisuke Yamamoto, Natsuyo Yamamoto, Ai Kotani, Kazuhiko Koike
Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities...
June 2, 2016: JCI Insight
Ao Huang, Xin Zhang, Shao-Lai Zhou, Ya Cao, Xiao-Wu Huang, Jia Fan, Xin-Rong Yang, Jian Zhou
PURPOSE: Circulating tumor DNA (ctDNA) is increasingly recognized as liquid biopsy to profile tumor genome. Droplet digital PCR (ddPCR) is a highly sensitive and easily operable platform for mutant detection. Here, we tried to detect ctDNA in hepatocellular carcinoma (HCC) patients using ddPCR. METHODS: Studies sequencing the genome of HCCs and COSMIC (Catalogue of Somatic Mutations in Cancer) database were reviewed to identify hotspot mutations. Circulating cell-free DNAs (cfDNAs) extracted from 1 ml preoperative plasma sample were analyzed to detect circulating mutants using ddPCR...
2016: Journal of Cancer
Yuta Aizawa, Akihide Koyama, Tomohiko Ishihara, Osamu Onodera, Akihiko Saitoh
BACKGROUND: Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients' clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated. OBJECTIVES: We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)-a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments-increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR...
September 23, 2016: Journal of Clinical Virology: the Official Publication of the Pan American Society for Clinical Virology
Xiaoli Xu, Cheng Peng, Xiaofu Wang, Xiaoyun Chen, Qiang Wang, Junfeng Xu
This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators...
September 8, 2016: Transgenic Research
Felix C K Wong, Kun Sun, Peiyong Jiang, Yvonne K Y Cheng, K C Allen Chan, Tak Y Leung, Rossa W K Chiu, Yuk Ming Dennis Lo
OBJECTIVES: The objectives of this study were to compare the concentrations, size profiles and major tissue contributors of cell-free DNA (cfDNA) in plasma and in serum. DESIGN AND METHODS: Thirteen pregnant women in the third trimester were recruited for this study. We collected EDTA-plasma and serum samples using various collection tubes. We determined their cfDNA concentrations and fetal cfDNA fractions using a zinc-finger X (ZFX)/zinc-finger Y (ZFY) droplet digital polymerase chain reaction (ZFX/ZFY ddPCR) assay...
September 9, 2016: Clinical Biochemistry
Ruurd C Verheul, Judith C T van Deutekom, Nicole A Datson
Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required...
2016: PloS One
Jeffrey D Palumbo, Teresa L O'Keeffe, Matthew W Fidelibus
Identification of populations of Aspergillus section Nigri species in environmental samples using traditional methods is laborious and impractical for large numbers of samples. We developed species-specific primers and probes for quantitative droplet digital PCR (ddPCR) to improve sample throughput and simultaneously detect multiple species in each sample. The ddPCR method was used to distinguish A. niger, A. welwitschiae, A. tubingensis and A. carbonarius in mixed samples of total DNA. Relative abundance of each species measured by ddPCR agreed with input ratios of template DNAs...
September 7, 2016: Letters in Applied Microbiology
Yacine Abed, Julie Carbonneau, Arnaud G L'Huillier, Laurent Kaiser, Guy Boivin
The H275Y and E119D neuraminidase (NA) mutations constitute important molecular markers of resistance to NA inhibitors in A(H1N1)pdm09 viruses. We used reverse transcriptase-droplet digital PCR amplification (RT-ddPCR) to analyze quasi-species at codons 275 and 119 of the NA in A(H1N1)pdm09 viruses recovered from an immuncompromised patient who received oseltamivir and zanamivir therapies. RT-ddPCR assays detected and quantified H275Y and E119D mutations with an efficiency that was comparable to that of high throughput sequencing (HiSeq 2500 Illumina) technology...
September 7, 2016: Journal of Medical Virology
Samantha L Schwartz, Anice C Lowen
Defective interfering (DI) particles are viruses that carry one or more large, internal deletions in the viral genome. These deletions occur commonly in RNA viruses due to polymerase error and yield incomplete genomes that typically lack essential coding regions. The presence of DI particles in a virus population can have a major impact on the efficiency of viral growth and is an important variable to consider in interpreting experimental results. Herein, we sought to develop a robust methodology for the quantification of DI particles within influenza A virus stocks...
November 2016: Journal of Virological Methods
Yang Zou, Peiwen Xu, Jie Li, Sexin Huang, Ming Gao, Ranran Kang, Xuan Gao, Yuan Gao
OBJECTIVE: To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. METHODS: A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively...
October 2016: Zhonghua Yi Xue Yi Chuan Xue za Zhi, Zhonghua Yixue Yichuanxue Zazhi, Chinese Journal of Medical Genetics
Y Y Kiseleva, K G Ptitsyn, S P Radko, V G Zgoda, A I Archakov
MicroRNA is a special type of regulatory molecules governing gene expression. Circulating microRNAs found in blood and other biological fluids are considered today as potential biomarkers of human pathology. Presently, quantitative alterations of particular microRNAs are revealed for a large number of oncological diseases and other disorders. The recently emerged method of digital droplet PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of human pathologies...
May 2016: Biomedit︠s︡inskai︠a︡ Khimii︠a︡
Anna Kristina Witte, Susanne Fister, Patrick Mester, Dagmar Schoder, Peter Rossmanith
Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora...
November 2016: Analytical and Bioanalytical Chemistry
Rekha Gyanchandani, Karthik J Kota, Amruth R Jonnalagadda, Tanya Minteer, Beth A Knapick, Steffi Oesterreich, Adam M Brufsky, Adrian V Lee, Shannon L Puhalla
ESR1 mutations are frequently acquired in hormone-resistant metastatic breast cancer (MBC). CDK4/6 inhibition along with endocrine therapy is a promising strategy in hormone receptor-positive MBC. However, the incidence and impact of ESR1 mutations on clinical outcome in patients treated with CDK4/6 inhibitors have not been defined. In this study, we evaluated the frequency of ESR1 mutations in cfDNA from 16 patients with MBC undergoing palbociclib and letrozole therapy. Four common ESR1 mutations (D538G, Y537C, Y537N, and Y537S) were analyzed in serial blood draws using ddPCR...
August 19, 2016: Oncotarget
Florence Nicot, Michelle Cazabat, Sébastien Lhomme, Olivier Marion, Karine Sauné, Julie Chiabrando, Martine Dubois, Nassim Kamar, Florence Abravanel, Jacques Izopet
The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar...
2016: Viruses
Takayuki Takahama, Kazuko Sakai, Masayuki Takeda, Koichi Azuma, Toyoaki Hida, Masataka Hirabayashi, Tetsuya Oguri, Hiroshi Tanaka, Noriyuki Ebi, Toshiyuki Sawa, Akihiro Bessho, Motoko Tachihara, Hiroaki Akamatsu, Shuji Bandoh, Daisuke Himeji, Tatsuo Ohira, Mototsugu Shimokawa, Yoichi Nakanishi, Kazuhiko Nakagawa, Kazuto Nishio
INTRODUCTION: Next-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to overcome resistance to earlier generations of such drugs mediated by a secondary T790M mutation of EGFR, but the performance of a second tumor biopsy to assess T790M mutation status can be problematic. METHODS: We developed and evaluated liquid biopsy assays for detection of TKI-sensitizing and T790M mutations of EGFR by droplet digital PCR (ddPCR) in EGFR mutation-positive non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance...
August 16, 2016: Oncotarget
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