Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane

Ma, Yu-Dong; Chang, Wen-Hsin; Luo, Kang; Wang, Chih‐Hung; Liu, Shih‐Yuan; Yen, Wen-Hsiang; Lee, Gwo‐Bin

doi:10.1016/j.bios.2017.08.026

Cited by 50 publications

(23 citation statements)

References 44 publications

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“…Additionally, the isothermal feature of our digital CRISPRbased detection assay confers faster amplification of the viral target using a simple constant-temperature heat bath, enabling rapid viral detection that can be deployed even in low-resource areas. In recent years, other digital isothermal methods, such as RPA-or LAMP-based digital PCR methods, have been developed for detecting a variety of DNA targets [49][50][51][52] . However, these methods are limited by their low specificity, due to the inherent tolerance of RPA/LAMP-based methods for base-pair mismatches as compared to traditional PCR methods [53][54][55][56][57][58] .…”

Section: Discussionmentioning

confidence: 99%

A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

Chan

Lee

et al. 2020

Preprint

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Since the outbreak of SARS-CoV-2, quantitative real-time PCR and CRISPR-based methods have been increasingly used for virus detection because of their speed (1 hour) and sensitivity. However, these methods do not allow for the absolute quantification of virus particles, which could reduce the inter-lab variability and accelerate the virus research. Although there is a digital PCR-based method that has been used for absolute virus quantification, its reaction time (4 hours) is too long for widespread application. Here, we report a rapid digital CRISPR method developed for the absolute quantification of SARS-CoV-2 DNA and Epstein−Barr virus DNA in human samples that yields results within 1 hour. We validated this method using synthetic SARS-CoV-2 DNA and Epstein−Barr viral DNA and compared results with those obtained with digital PCR. Digital CRISPR allows absolute quantification of DNA with a dynamic range from 0.6 to 2027 copies/µL (R2 value > 0.98), without cross reactivity on similar virus and human background DNA. Thus, our digital CRISPR can accurately detect and quantify nucleic acid in 1h without thermal cycling, which provides a 4-fold faster alternative to digital PCR-based virus detection.

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Section: Discussionmentioning

confidence: 99%

A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

Chan

Lee

et al. 2020

Preprint

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“…This approach avoids the risk of losing the sample through a small leak. PDMS presents however several drawbacks: (1) DNA and protein tends to absorb onto its hydrophobic surface if not properly pre-incubated with a solution of BSA [ 67 , 73 ], which in turn may affect its surface properties; (2) it is permeable to water and evaporation must be mitigated by incorporating water reservoirs [ 74 ] and vapor barriers made of parylene C [ 37 ] or glass [ 43 ], which complicates device fabrication; (3) it suffers from a high cost of production, which impedes its use in large-scale manufacturing.…”

Section: Miniaturization and Hyper-compartmentalizationmentioning

confidence: 99%

dPCR: A Technology Review

Quan

Sauzade

Brouzés

2018

Sensors

472

350

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Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

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“…Rane and colleagues recently reported an integrated-microfluidic system for digital nucleic acid detection through droplet generation, incubation and in-line detection for digital LAMP within a single device [49]. Also, a hydrophilic PDMS that allows LAMP to be performed in a self-driven DMF device has been announced [50]. For the detection of influenza A virus [51] and Zika virus [52], reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) has also been merged with DMF technologies.…”

Section: Droplet-based Microfluidics For Genomicsmentioning

confidence: 99%

Recent Advances in Droplet-based Microfluidic Technologies for Biochemistry and Molecular Biology

2019

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Recently, droplet-based microfluidic systems have been widely used in various biochemical and molecular biological assays. Since this platform technique allows manipulation of large amounts of data and also provides absolute accuracy in comparison to conventional bioanalytical approaches, over the last decade a range of basic biochemical and molecular biological operations have been transferred to drop-based microfluidic formats. In this review, we introduce recent advances and examples of droplet-based microfluidic techniques that have been applied in biochemistry and molecular biology research including genomics, proteomics and cellomics. Their advantages and weaknesses in various applications are also comprehensively discussed here. The purpose of this review is to provide a new point of view and current status in droplet-based microfluidics to biochemists and molecular biologists. We hope that this review will accelerate communications between researchers who are working in droplet-based microfluidics, biochemistry and molecular biology.

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Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane

Cited by 50 publications

References 44 publications

A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

dPCR: A Technology Review

Recent Advances in Droplet-based Microfluidic Technologies for Biochemistry and Molecular Biology

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