Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Three-color crystal digital PCR.

ABSTRACT: Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.

SUBMITTER: Madic J

PROVIDER: S-EPMC5154636 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Three-color crystal digital PCR.

Madic J J Zocevic A A Senlis V V Fradet E E Andre B B Muller S S Dangla R R Droniou M E ME

Biomolecular detection and quantification 20161103

Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel tech ...[more]

PMID: 27990348

Similar Datasets

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

Project description:We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring.

| S-EPMC6593258 | biostudies-literature

Digital quantification of human eye color highlights genetic association of three new loci.

Project description:Previous studies have successfully identified genetic variants in several genes associated with human iris (eye) color; however, they all used simplified categorical trait information. Here, we quantified continuous eye color variation into hue and saturation values using high-resolution digital full-eye photographs and conducted a genome-wide association study on 5,951 Dutch Europeans from the Rotterdam Study. Three new regions, 1q42.3, 17q25.3, and 21q22.13, were highlighted meeting the criterion for genome-wide statistically significant association. The latter two loci were replicated in 2,261 individuals from the UK and in 1,282 from Australia. The LYST gene at 1q42.3 and the DSCR9 gene at 21q22.13 serve as promising functional candidates. A model for predicting quantitative eye colors explained over 50% of trait variance in the Rotterdam Study. Over all our data exemplify that fine phenotyping is a useful strategy for finding genes involved in human complex traits.

| S-EPMC2865509 | biostudies-literature

Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion.

Project description:The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

| S-EPMC7568307 | biostudies-literature

Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus.

Project description:Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. APPV distributes throughout the world and causes certain economic losses to the swine industry. The specific primers and probe were designed targeting the 5' untranslated region (UTR) of APPV to amplify a 90 bp fragment, and the recombinant standard plasmid was constructed. After optimizing the concentrations of primers and probe, annealing temperature, and reaction cycles, a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) were successfully established. The results showed that the standard curves of the qRT-PCR and the cdRT-PCR had R2 values of 0.999 and 0.9998, respectively. Both methods could specifically detect APPV, and no amplification signal was obtained from other swine viruses. The limit of detection (LOD) of the cdRT-PCR was 0.1 copies/µL, and that of the qRT-PCR was 10 copies/µL. The intra-assay and inter-assay coefficients of variation of repeatability and reproducibility were less than 0.90% for the qRT-PCR and less than 5.27% for the cdRT-PCR. The 60 clinical tissue samples were analyzed using both methods, and the positivity rates of APPV were 23.33% by the qRT-PCR and 25% by the cdRT-PCR, with a coincidence rate of 98.33%. The results indicated that the cdRT-PCR and the qRT-PCR developed here are highly specific, sensitive methods for the rapid and accurate detection of APPV.

| S-EPMC10223196 | biostudies-literature

Digital color in cellulose nanocrystal films.

Project description:Cellulose nanocrystals (CNCs) form chiral nematic phases in aqueous suspensions that can be preserved upon evaporation of water. The resulting films show an intense directional coloration determined by their microstructure. Here, microreflection experiments correlated with analysis of the helicoidal nanostructure of the films reveal that the iridescent colors and the ordering of the individual nematic layers are strongly dependent on the polydispersity of the size distribution of the CNCs. We show how this affects the self-assembly process, and hence multidomain color formation in such bioinspired structural films.

| S-EPMC4251880 | biostudies-other

Digital Computer Matching of Tooth Color

Project description:This study aimed to determine the validity of the digital photocolorimetric (PCM) method in matching the color of human teeth. First, two Vitapan Classical shade guides, each containing 16 shade guide teeth, were visually shade matched, and digital photographs of each three pair of shade guide teeth were taken in a color matching booth. Secondly, visual shade matching of the upper central incisors of 48 subjects was performed by two prosthodontists independently in a chair, using the Vitapan Classical shade guide. The three closest shade guide teeth were visually selected and ranked in order of preference, for which digital photographs were taken under ceiling daylight-corrected fluorescent lighting. All digital images were analyzed on a computer screen using software to calculate the color difference between the reference tooth and other teeth in the same digital image. The percent color matching for the shade guide teeth and human teeth was 88% and 75%, respectively. There was no statistically significant difference in matching the tooth color between the shade guide teeth and human teeth. The digital PCM method is valid for the range of human teeth based on the Vitapan Classical shade guide. This method enhances communication with the laboratory personnel in matching the tooth color.

| S-EPMC5521749 | biostudies-literature

High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.

Project description:In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.

| S-EPMC3982983 | biostudies-literature

Plasma-based early screening and monitoring of EGFR mutations in NSCLC patients by a 3-color digital PCR assay.

Project description:BackgroundNoninvasive plasma-based detection of EGFR mutations using digital PCR promises a fast, sensitive and reliable approach to predicting the efficiency of EGFR-TKI. However, the low throughput and high cost of digital PCR restricts its clinical application.MethodsWe designed a digital PCR assay, which can simultaneously detect 39 mutations of exons 18-21 of the EGFR gene. To assess overall performance, retrospective FFPE tissues from 30 NSCLC patients and plasma from 33 NSCLC patients were collected and analysed.ResultsThe LoD of the EGFR mutations was as low as 0.308 copies/μL, and the linear correlation between the detected and expected values at different concentrations (0.01-10%) was low as well. Compared to ARMS-PCR in FFPE, the accuracy values of the dEGFR39 assay in plasma from 33 patients was 87.88% (29/33, 95% CI 72.67-95.18%). While monitoring the 33 patients, the EGFR mutation load as assessed by dEGFR39 was associated with the objective response to treatment. Thirteen samples from eight patients were identified by dEGFR39 to harbour the T790M mutation over time; of these patients, only nine (69%) were detected using SuperARMS.ConclusionOur results indicate that dEGFR39 assay is reliable, sensitive and cost-efficient. This method is beneficial for profiling EGFR mutations for precision therapy and prognosis after TKI treatment, especially in patients with insufficient tissue biopsy samples.

| S-EPMC7592053 | biostudies-literature

Color restoration based on digital pathology image.

Project description:ObjectiveProtective color restoration of faded digital pathology images based on color transfer algorithm.MethodsTwenty fresh tissue samples of invasive breast cancer from the pathology department of Qingdao Central Hospital in 2021 were screened. After HE staining, HE stained sections were irradiated with sunlight to simulate natural fading, and every 7 days was a fading cycle, and a total of 8 cycles were experienced. At the end of each cycle, the sections were digitally scanned to retain clear images, and the color changes of the sections during the fading process were recorded. The color transfer algorithm was applied to restore the color of the faded images; Adobe Lightroom Classic software presented the histogram of the image color distribution; UNet++ cell recognition segmentation model was used to identify the color restored images; Natural Image Quality Evaluator (NIQE), Information Entropy (Entropy), and Average Gradient (AG) were applied to evaluate the quality of the restored images.ResultsThe restored image color met the diagnostic needs of pathologists. Compared with the faded images, the NIQE value decreased (P<0.05), Entropy value increased (P<0.01), and AG value increased (P<0.01). The cell recognition rate of the restored image was significantly improved.ConclusionThe color transfer algorithm can effectively repair faded pathology images, restore the color contrast between nucleus and cytoplasm, improve the image quality, meet the diagnostic needs and improve the cell recognition rate of the deep learning model.

| S-EPMC10306179 | biostudies-literature

Comparative evaluation of three commercial quantitative cytomegalovirus standards by use of digital and real-time PCR.

Project description:The recent development of the 1st WHO International Standard for human cytomegalovirus (CMV) and the introduction of commercially produced secondary standards have raised hopes of improved agreement among laboratories performing quantitative PCR for CMV. However, data to evaluate the trueness and uniformity of secondary standards and the consistency of results achieved when these materials are run on various assays are lacking. Three concentrations of each of the three commercially prepared secondary CMV standards were tested in quadruplicate by three real-time and two digital PCR methods. The mean results were compared in a pairwise fashion with nominal values provided by each manufacturer. The agreement of results among all methods for each sample and for like concentrations of each standard was also assessed. The relationship between the nominal values of standards and the measured values varied, depending upon the assay used and the manufacturer of the standards, with the degree of bias ranging from +0.6 to -1.0 log10 IU/ml. The mean digital PCR result differed significantly among the secondary standards, as did the results of the real-time PCRs, particularly when plotted against nominal log10 IU values. Commercially available quantitative secondary CMV standards produce variable results when tested by different real-time and digital PCR assays, with various magnitudes of bias compared to nominal values. These findings suggest that the use of such materials may not achieve the intended uniformity among laboratories measuring CMV viral load, as envisioned by adaptation of the WHO standard.

| S-EPMC4400765 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data