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      Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR

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          Abstract

          Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes Δ prfA, showed even slightly better quantification over a broader dynamic range.

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          Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA

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          The online version of this article (doi:10.1007/s00216-016-9861-9) contains supplementary material, which is available to authorized users.

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          Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances.

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            Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

            An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass.
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              Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.

              Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log(10) versus 4 log(10) copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R(2) ≥ 0.98 in each of 6 regression models) and clinical samples (R(2) = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.
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                Author and article information

                Contributors
                peter.rossmanith@vetmeduni.ac.at
                Journal
                Anal Bioanal Chem
                Anal Bioanal Chem
                Analytical and Bioanalytical Chemistry
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                1618-2642
                1618-2650
                24 August 2016
                24 August 2016
                2016
                : 408
                : 27
                : 7583-7593
                Affiliations
                [1 ]Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria
                [2 ]Institute of Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria
                Article
                9861
                10.1007/s00216-016-9861-9
                5061835
                27558101
                e1137e6b-13ff-4425-8d95-278179ee28f3
                © The Author(s) 2016

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 19 June 2016
                : 3 July 2016
                : 11 July 2016
                Categories
                Paper in Forefront
                Custom metadata
                © Springer-Verlag Berlin Heidelberg 2016

                Analytical chemistry
                ddpcr,qpcr,prfa,listeria monocytogenes,poisson distribution,heterogeneous matrix
                Analytical chemistry
                ddpcr, qpcr, prfa, listeria monocytogenes, poisson distribution, heterogeneous matrix

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