Molecular quantification of environmental DNA using microfluidics and digital PCR

Hoshino, Takao; Inagaki, Fumio

doi:10.1016/j.syapm.2012.06.006

Cited by 112 publications

(105 citation statements)

References 27 publications

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“…dPCR is relatively tolerant to inhibitors compared to qPCR (35 ). dPCR is not dependent on amplification curves that may be affected by subtle inhibitors as in qPCR.…”

Section: Reduced Susceptibility To Inhibitorsmentioning

confidence: 99%

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Huggett¹,

Foy²,

Beneš³

et al. 2013

Clinical Chemistry

746

660

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There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

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“…dPCR is relatively tolerant to inhibitors compared to qPCR (35 ). dPCR is not dependent on amplification curves that may be affected by subtle inhibitors as in qPCR.…”

Section: Reduced Susceptibility To Inhibitorsmentioning

confidence: 99%

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Huggett¹,

Foy²,

Beneš³

et al. 2013

Clinical Chemistry

746

660

View full text Add to dashboard Cite

show abstract

“…Inhibition of dPCR appears to cause less negative bias than with qPCR (31,37,38 ); however, inhibitors are able to reduce the quantitative value measured by dPCR while maintaining good precision (31 ), and we strongly recommend including internal positive controls when measuring clinical samples, especially if negative results need to be reported. The popular routine use of internal positive controls when applying qPCR clinically is equally important for dPCR.…”

Section: Continued On Page 83mentioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

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“…The abundance of the whole prokaryotic 16S rRNA genes with depth showed similar values and profiles to the microbial cell abundance in all of the holes. However, several shallow sediment depths in Holes C0014B and C0014G exhibited large data gaps between the microbial cell abundance and the whole prokaryotic 16S rRNA gene abundance due to the relatively higher detection limit of the microbial cell count, as shown in Figure 1c, or due to the technical limitations of the Q-PCR (Hoshino and Inagaki, 2012;Lloyd et al, 2013;Morono et al, 2014). The relative abundance of archaeal 16S rRNA genes increased with depth and represented approximately half of the whole prokaryotic 16S rRNA gene assemblages in the deepest zones where Q-PCR could detect 16S rRNA genes (Supplementary Figure S3).…”

Section: Geochemical Evidence Of Hydrothermal Fluid Input and Methanementioning

confidence: 99%

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor

et al. 2016

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Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the MidOkinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ 13 C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to~45°C. However, in the past, higher temperatures of 106-198°C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

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Molecular quantification of environmental DNA using microfluidics and digital PCR

Cited by 112 publications

References 27 publications

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor

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