PCR digitale en micro-compartiments

Perez‐Toralla, Karla; Pekin, Deniz; Bartolo, Jean-François; Garlan, Fanny; Nizard, Philippe; Laurent‐Puig, Pierre; Taly, Valérie

doi:10.1051/medsci/20153101017

Cited by 8 publications

(2 citation statements)

References 41 publications

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“…One of the most attractive fields of dPCR application is cancer research [ 8 , 9 ]. Due to its ability to quantify small amounts of mutated DNA molecules (MUT) among a large number of wild-type molecules (WT, non-mutated), this technique permits the detection of rare or low abundant alleles in cancer patient samples [ 10 , 11 ]. In particular, dPCR allows the detection of mutations in circulating tumor DNA from liquid biopsy (e.g.…”

Section: Introductionmentioning

confidence: 99%

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

et al. 2016

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In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

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Section: Introductionmentioning

confidence: 99%

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

et al. 2016

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“…Currently, tremendous research efforts have been made to achieve the simple, rapid, cost-effective, and reliable compartmentalization for the digital nucleic acid amplification and analysis − using several methods, including solid microchambers/wells, water-in-oil emulsion, and printing. ,− However, these methods suffer from technical issues, such as difficulties in finding the optimal surfactant-to-oil ratio and the requirement of sophisticated micro-/nanofabrication facilities. Hence, microcontact printing (μCP), a form of soft lithography based on an elastomer stamp, is employed as an alternative approach for producing accurate, reproducible, and reliable sessile microdroplet arrays without the need for multiple cleanroom steps, enabling simple and rapid micropatterning. ,, Based on these advantageous features, μCP is widely utilized for diverse biological applications, such as protein and cell analysis. − …”

Section: Introductionmentioning

confidence: 99%

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection

Choi

Song

Kim

et al. 2021

ACS Appl. Mater. Interfaces

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The digital nucleic acid assay is a precise, sensitive, and reproducible method for determining the presence of individual target molecules separated in designated partitions; thus, this technique can be used for the nucleic acid detection. Here, we propose a multifunctional micropattern array capable of isolating individual target molecules into partitions and simultaneous on-site cell lysis to achieve a direct DNA extraction and digitized quantification thereof. The multifunctional micropattern array is fabricated by the deposition of a copolymer film, poly(2-dimethylaminomethyl styrene-co-hydroxyethyl methacrylate) (pDH), directly on a microfluidic chip surface via the photoinitiated chemical vapor deposition process, followed by hydrophobic microcontact printing (μCP) to define each partition for the nucleic acid isolation. The pDH layer is a positively charged surface, which is desirable for the bacterial lysis and DNA capture, while showing exceptional water stability for more than 24 h. The hydrophobic μCP-treated pDH surface is stable under aqueous conditions at a high temperature (70 °C) for 1 h and enables the rapid and reliable formation of thousands of sessile microdroplets for the compartmentalization of an aqueous sample solution without involving bulky and costly microfluidic devices. By assembling the multifunctional micropattern array into the microfluidic chip, the isothermal amplification in each partition can detect DNA templates over a concentration range of 0.01–2 ng/μL. The untreated bacterial cells can also be directly compartmentalized via the microdroplet formation, followed by the on-site cell lysis and DNA capture on the compartmentalized pDH surface. For Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus cells, cell numbers ranging from 1.4 × 104 to 1.4 × 107 can be distinguished by using the multifunctional micropattern array, regardless of the cell type. The multifunctional micropattern array developed in this study provides a novel multifunctional compartmentalization method for rapid, simple, and accurate digital nucleic acid assays.

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Current, Emerging, and Future Applications of Digital PCR in Non-Invasive Prenatal Diagnosis

Nectoux

2017

Mol Diagn Ther

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Digital PCR (dPCR) approaches have been developed for the detection of nucleic acids of low abundance, such as cell-free DNA, and represent an attractive and sensitive alternative to conventional methods, particularly in the field of non-invasive prenatal diagnosis (NIPD). In this review, we present the principle of dPCR and its applications in the field of prenatal diagnosis from current and emerging uses, such as fetal gender determination, rhesus blood group D antigen genotyping, or monogenic disorders prenatal testing, to future applications, such as the diagnosis and monitoring of pregnancy-related disorders. We also address considerations for implementation of the method in a clinical laboratory and discuss the competiveness of dPCR over other technologies such as quantitative PCR or massively parallel sequencing.

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PCR digitale en micro-compartiments

Cited by 8 publications

References 41 publications

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection

Current, Emerging, and Future Applications of Digital PCR in Non-Invasive Prenatal Diagnosis

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