Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment

Cao, Yiping; Raith, Meredith R.; Griffith, John F.

doi:10.1016/j.watres.2014.12.008

Cited by 154 publications

(183 citation statements)

References 24 publications

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“…Most previous studies have used ddPCR to amplify low concentrations of target sequences, such as when detecting endangered species or fecal contamination in water samples, but many rely upon artificially spiking predetermined amounts of purified target DNA into reactions to determine the utility of ddPCR and compare it to real-time PCR (e.g., [27, 28, 24]). This assay is one of the first ddPCR applications capable of detecting rare sequences in moth samples collected from field traps, and not simply in laboratory simulations.…”

Section: Discussionmentioning

confidence: 99%

“…Many ddPCR studies have adapted real-time PCR methods to determine the functionality of ddPCR in previously established assays [27, 28, 29, 30]. For example, ddPCR has been used to observe fecal contamination and the presence of invasive species in environmental DNA samples, in the detection of GMO plant specimens, and to determine the prevalence of rare mutations in circulating, cell-free DNA in patient blood samples [27, 28, 30, 24].…”

Section: Introductionmentioning

confidence: 99%

“…For example, ddPCR has been used to observe fecal contamination and the presence of invasive species in environmental DNA samples, in the detection of GMO plant specimens, and to determine the prevalence of rare mutations in circulating, cell-free DNA in patient blood samples [27, 28, 30, 24]. These types of studies set a precedent for our experiments, as they represent varying situations in which small amounts of target DNA are identified within a large background of non-target DNA.…”

Section: Introductionmentioning

confidence: 99%

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A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

et al. 2017

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Moths in the genus Helicoverpa are some of the most important agricultural pests in the world. Two species, H. armigera (Hübner) and H. zea (Boddie), cause the majority of damage to crops and millions of dollars are spent annually on control of these pests. The recent introduction of H. armigera into the New World has prompted extensive survey efforts for this species in the United States. Surveys are conducted using bucket traps baited with H. armigera pheromone, and, because the same pheromone compounds attract both species, these traps often capture large numbers of the native H. zea. Adult H. armigera and H. zea are very similar and can only be separated morphologically by minor differences in the genitalia. Thus, a time consuming genitalic dissection by a trained specialist is necessary to reliably identify either species, and every specimen must be dissected. Several molecular methods are available for differentiating and identifying H. armigera and H. zea, including two recently developed rapid protocols using real-time PCR. However, none of the published methods are capable of screening specimens in large batches. Here we detail a droplet digital PCR (ddPCR) assay that is capable of detecting a single H. armigera in a background of up to 999 H. zea. The assay has been tested using bulk extractions of 1,000 legs from actual trap samples and is effective even when using poor quality samples. This study provides an efficient, rapid, reproducible, and scalable method for processing H. armigera survey trap samples in the U.S. and demonstrates the potential for applying ddPCR technology to screen and diagnose invasive species.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

et al. 2017

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“…The ratio between positive partitions and all counted partitions is used in the calculation of the initial target concentration, with use of the Poisson distribution [38]. This technology is being widely adopted for absolute quantification in different areas of research and diagnostics [39–48], including in the field of GMOs, which is further described and discussed in the following text.…”

Section: Overview Of Different Technologies Used For Gmo Detectionmentioning

confidence: 99%

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Demeke¹,

Dobnik

2018

Anal Bioanal Chem

110

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The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing. Graphical abstractThere are generally three different options for absolute quantification of genetically modified organisms (GMOs) using digital PCR: droplet- or chamber-based and droplets in chambers. All have in common the distribution of reaction mixture into several partitions, which are all subjected to PCR and scored at the end-point as positive or negative. Based on these results GMO content can be calculated.

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“…First introduced in the 1990s [3], [4], dPCR is increasingly being utilised for quantification of DNA targets and rare events [2], [5], [6], pathogen detection [7], [8], viral load testing [9], [10], detection of genetically modified organisms in food [11] and quantification of massively parallel sequence libraries [12]. More recently, an international comparison study [13] conducted between metrology institutes demonstrated excellent comparability between dPCR and qPCR results, and, in general, dPCR resulted in better precision.…”

mentioning

confidence: 99%

Digital polymerase chain reaction for characterisation of DNA reference materials

Bhat

Emslie

2016

Biomolecular Detection and Quantification

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Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard. Due to these properties, this technique has the potential to not only improve routine quantitative nucleic acid analysis, but also to be used as a reference method for certification of nucleic acid RM. The article focuses on the use and application of both dPCR and RMs for accurate quantification.

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Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment

Cited by 154 publications

References 24 publications

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

A droplet digital PCR (ddPCR) assay to detect Helicoverpa armigera (Lepidoptera: Noctuidae) in bulk trap samples

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

Digital polymerase chain reaction for characterisation of DNA reference materials

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