A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

Witte, Anna Kristina; Mester, Patrick; Fister, Susanne; Witte, Matthias; Schoder, Dagmar; Rossmanith, Peter

doi:10.1371/journal.pone.0168179

Cited by 54 publications

(63 citation statements)

References 27 publications

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“…2 , Supplemental Table S1): Firstly, there are severe differences between the polymerases in terms of qPCR performance, and secondly, the effect is much more distinct in the prfA assay than in the Δ prfA assay. The latter effect is in agreement with data obtained in ddPCR, where the Δ prfA assay was less sensitive to assay modifications than the prfA assay [ 12 ]. In the prfA assay, only three of the ten polymerases/mastermixes amplified the complete DNA range of the calibration curve, while eight of the ten succeeded in the Δ prfA assay.…”

Section: Resultssupporting

confidence: 91%

“…Moreover, the prfA assay has been tested and optimized for droplet digital PCR (ddPCR). For this application, the assay requires different amplification conditions, which have been comprehensively studied [ 6 , 12 ]. ddPCR is a relatively new PCR method based on Poisson distribution and permits quantification without external standards [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

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Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

Witte

Sickha

Mester

et al. 2018

Biomolecular Detection and Quantification

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The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

Witte

Sickha

Mester

et al. 2018

Biomolecular Detection and Quantification

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“…Each experimental sample included 0.5 µL of the 50 µL re-suspended eDNA, using 5 µL of a 1/10 dilution series to reduce pipetting error. This volume of the extracted eDNA was chosen after initial "quenching experiments" showed evidence of inhibitors in conventional PCR experiments and initial ddPCR reactions showed evidence of droplet "rain" (Witte et al, 2016). The positive control was 1 µL of a 1/1,000 dilution of total cellular DNA extracted from a skin biopsy sample of a Hector's dolphin, Cephalorhynchus hectori hectori (Hamner et al, 2012).…”

Section: Digital Droplet (Dd)pcrmentioning

confidence: 99%

Environmental DNA (eDNA) From the Wake of the Whales: Droplet Digital PCR for Detection and Species Identification

et al. 2018

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Genetic sampling for identification of species, subspecies or stock of whales, dolphins and porpoises at sea remains challenging. Most samples have been collected with some form of a biopsy dart requiring a close approach of a vessel while the individual is at the surface. Here we have adopted droplet digital (dd)PCR technology for detection and species identification of cetaceans using environmental (e)DNA collected from seawater. We conducted a series of eDNA sampling experiments during 25 encounters with killer whales, Orcinus orca, in Puget Sound (the Salish Sea). The regular habits of killer whales in these inshore waters allowed us to locate pods and collect seawater, at an initial distance of 200 m and at 15-min intervals, for up to 2 h after the passage of the whales. To optimize detection, we designed a set of oligonucleotide primers and probes to target short fragments of the mitochondrial (mt)DNA control region, with a focus on identification of known killer whale ecotypes. We confirmed the potential to detect eDNA in the wake of the whales for up to 2 h, despite movement of the water mass by several kilometers due to tidal currents. Re-amplification and sequencing of the eDNA barcode confirmed that the ddPCR detection included the "southern resident community" of killer whales, consistent with the calls from hydrophone recordings and visual observations.

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“…Third-generation PCR, known as a droplet digital PCR (ddPCR), allows fast and quantitative detection (Cremonesi et al 2016). Droplet digital PCR, based on the amplification of a single target DNA molecule in a plurality of separate droplets, enables an exact quantification of DNA copy numbers (Witte et al 2016b). The principle of ddPCR method is founded on DNA analysis according to the Poisson distribution.…”

Section: Introductionmentioning

confidence: 99%

Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR

2020

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Listeria monocytogenes is a Gram-positive bacterium, commonly found in food, water or sewage. This microorganism is capable of forming biofilm on different surfaces such as steel, glass, polypropylene etc. Recently an increase in cases of listeriosis has been noted, making L. monocytogenes the important health threat. Therefore, there is a need for rapid and sensitive detection of this pathogen. This study aimed to compare the number of L. monocytogenes cells recovered from the biofilm (prepared on steel and polypropylene) using the detection and amplification of the hlyA gene (droplet digital PCR, ddPCR) and the classical culture method. The research material consisted of 96 L. monocytogenes strains. A total of 58 isolates were obtained from clinical samples and 38 isolates derived from the municipal sewage treatment plant. Additionally, the reference strain ATCC ® 19111 ™ (WDCM00020) was used. The Pearson correlation coefficient for the results obtained by the classical culture-based method and ddPCR was 0.864 and 0.725, for biofilms produced on AISI 304 stainless steel surface and the polypropylene surface, respectively. Correlations were statistically significant (p ≤ 0.001), indicating that the ddPCR technique is an effective tool for the assessment of bacteria number in the biofilm.

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A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

Cited by 54 publications

References 27 publications

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

Essential role of polymerases for assay performance – Impact of polymerase replacement in a well-established assay

Environmental DNA (eDNA) From the Wake of the Whales: Droplet Digital PCR for Detection and Species Identification

Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR

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