Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples

Rački, Nejc; Dreo, T.; Gutiérrez-Aguirre, Ion; Blejec, Andrej; Ravnikar, Maja

doi:10.1186/s13007-014-0042-6

Cited by 202 publications

(138 citation statements)

References 27 publications

(37 reference statements)

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“…Additional experiments from other scientific fields than diagnostic virology are in line with these findings, mostly showing a decreased susceptibility to inhibition in dPCR [40] and RT-dPCR experiments [23,41,42].…”

Section: Pcr Inhibitory Substancessupporting

confidence: 79%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

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Section: Pcr Inhibitory Substancessupporting

confidence: 79%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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“…Similarly, much of the variability seen in the present study was present at lower target concentrations. Others, however, have supported the advantages of dPCR in reducing susceptibility to PCR inhibitors (13)(14)(15). Here and elsewhere, when using a common platform, results have been similar or identical irrespective of the reagents used.…”

Section: Discussionmentioning

confidence: 83%

“…Based on the principles of limiting dilution or partition, together with endpoint PCR, digital methods remove dependence on rate-based quantitation (10)(11)(12). They are therefore potentially less sensitive to the presence of PCR inhibitors or other sources of variation in assay efficiency (13)(14)(15), and they no longer require the use of a calibration curve to produce quantitative data. As such, it might be expected that accuracy and interassay agreement will improve over those seen with real-time methods.…”

mentioning

confidence: 99%

Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR

Hayden

Sam

et al. 2016

J Clin Microbiol

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A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples (n ‫؍‬ 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results. The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another. When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two. Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads. Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values.

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“…Thus, dPCR possesses the following potential advantages over qPCR. dPCR can be used to accurately determine the number of nucleic acid molecules in a sample without certified reference material and a standard curve; the dilution of a template also correspondingly dilutes inhibitors possibly present in the template to make dPCR less sensitive to inhibitors [27] [28], therefore further improving accuracy and efficiency. These properties and advantages make dPCR an excellent tool for many applications where sensitivity or precise quantification of nucleic acids is needed, such as identifying mutations or copy number variations in tumor cells, detection of low copy number nucleic acid targets, or examining gene expression at the single-cell level [26] -digital-pcr-system.html).…”

Section: Introductionmentioning

confidence: 99%

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

Wan¹,

Li²,

Wu³

et al. 2016

ABB

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Detection and quantification of transgenes are important in analyzing genetically modified organisms (GMOs). Quantitative polymerase chain reaction (qPCR) is commonly utilized for such purposes. However, qPCR has certain limitations in detecting and quantifying transgenes in GMOs, such as the need of certified reference materials, a standard curve, and possible affection by inhibitors. Therefore, alternative and possibly better methods are needed. Recent advances in digital PCR technologies have promised to allow accurate quantification of nucleic acids and therefore provided another useful technique to analyze GMOs. Thermo Fisher Scientific has recently commercialized the Applied Biosystems QuantStudio 3D digital PCR system that can be used for a wide range of applications involving nucleic acids. It will be beneficial to the scientific community to show the applicability of this digital PCR system in detecting and quantifying transgenes in GMOs. In the present study, the transgenes present in the Roundup® Ready Soybean (RR1, event 40-3-2) and Roundup Ready Soybean 2 (RR2, event MON89788) developed by Monsanto Corporation were analyzed by using this digital PCR system. The qPCR analysis results were included for comparison. Using specifically designed TaqMan assays, as low as 1% of the RR1 or RR2 soybean material was reliably detected and quantified on the dPCR platform. Therefore, digital PCR is a sensitive and reliable method to analyze the RR transgenic soybeans, and should be another useful tool for analyzing other transgenic plants.

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Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples

Cited by 202 publications

References 27 publications

The Future of Digital Polymerase Chain Reaction in Virology

The Future of Digital Polymerase Chain Reaction in Virology

Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR

Application of Digital PCR in the Analysis of Transgenic Soybean Plants

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