Droplet digital PCR technology promises new applications and research areas

Manoj, P.

doi:10.3109/19401736.2014.913168

Cited by 41 publications

(32 citation statements)

References 22 publications

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“…In addition, the signals obtained from these droplets could be recognized as false signals due to the abnormally high fluorescence intensity measured in ddPCR assay [21,22]. Nevertheless, the difference in the average number of VIC dyes detected between the Hanwoo and Hol- In ddPCR assay, DNA is divided into numerous wells or droplets, and the concentration of target region is absolute quantified using Poisson statistics [23,24]. The ddPCR assay can be quantified with high accuracy in counting single molecules and analyzing a small number of copies of a particular population [25,26].…”

Section: Resultsmentioning

confidence: 99%

Quantitative evaluation of the molecular marker using droplet digital PCR

Shin

Kim

Oh³

et al. 2020

Genomics Inform

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Transposable elements (TEs) constitute approximately half of Bovine genome. They can be a powerful species-specific marker without regression mutations by the structure variation (SV) at the time of genomic evolution. In a previous study, we identified the Hanwoo-specific SV that was generated by a TE-association deletion event using traditional PCR method and Sanger sequencing validation. It could be used as a molecular marker to distinguish different cattle breeds (i.e., Hanwoo vs. Holstein). However, PCR is defective with various final copy quantifications from every sample. Thus, we applied to the droplet digital PCR (ddPCR) platform for accurate quantitative detection of the Hanwoo-specific SV. Although samples have low allele frequency variation within Hanwoo population, ddPCR could perform high sensitive detection with absolute quantification. We aimed to use ddPCR for more accurate quantification than PCR. We suggest that the ddPCR platform is applicable for the quantitative evaluation of molecular markers.

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Section: Resultsmentioning

confidence: 99%

Quantitative evaluation of the molecular marker using droplet digital PCR

Shin

Kim

Oh³

et al. 2020

Genomics Inform

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“…As a result, each reaction contains one or no copy of nucleotide sequences of interest that can be assayed individually and counted without requiring a standard curve and correcting by Poisson statistics (Baker 2012). Compared to qPCR, dPCR is more advantageous in terms of its sensitivity, specificity and precision (Baker 2012;Köppel et al 2019;Manoj 2016;Ren et al 2017;Cao et al 2020, Wang et al 2018.…”

Section: Introductionmentioning

confidence: 99%

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration

Temisak¹,

Thangsunan²,

Boonil³

et al. 2020

Preprint

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The problem in meat adulteration and food fraud emphasised the requirement of developing accurate analytical approaches for the quantitative detection in helping the control of meat adulteration. In this study, the droplet digital Polymerase Chain Reaction (ddPCR) assays to quantify the ratios of pork DNA to the total amount of meat DNA were developed by challenging against DNA extracted from a range of gravimetrically prepared matrices of pork in beef. A single copy nuclear DNA gene, β-actin, was employed as a target gene, accompanied with myostatin gene as a cross species target for mammal and poultry meat background in order to quantifying approach. All the developed assays, singleplex, duplex and triplex did not show significant difference in quantification of pork content in beef background and demonstrated a good and comparable performance to the mass fractions. The singleplex assay provided more biases than the other two assays when performing with a low concentration of target species. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and running time. All proposed methods allowed us to quantify pork addition in beef with a limit of quantification (LOQ) estimated at 0.1% (w/w) and a limit of detection (LOD) down to 0.01% (w/w). The developed triplex assay was also tested with commercial processed foods and showed the ability to determine not only the presence of particular pork or beef but also the quantitative purpose directly without standard curves. Hence, the developed ddPCR assays demonstrated a good trueness and precision of the methods in quantifying pork or beef content for meat adulteration. It is expected that these developed approaches can be applied to help regulators to confidently enforce food labelling obligations.

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“…6 Unlike qPCR, ddPCR uses an end-point approach, with samples defined as “positive” when a fluorescent signal exceeds a user-defined threshold. During partitioning, template gDNA is randomly distributed within volumetrically defined water-in-oil sample droplets.…”

Section: Introductionmentioning

confidence: 99%

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Lin

Okumura

Yatsuda³

et al. 2016

Human Gene Therapy Methods

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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. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

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Droplet digital PCR technology promises new applications and research areas

Cited by 41 publications

References 22 publications

Quantitative evaluation of the molecular marker using droplet digital PCR

Quantitative evaluation of the molecular marker using droplet digital PCR

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

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