Digital PCR absolute quantification of a DNA plasmid based on enzyme digestion

2015 ◽  
pp. 913-916
Author(s):  
Wen Liang ◽  
Li Xu ◽  
Yan Li ◽  
YanLi Wen ◽  
Lanying Li ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Enzyme Digestion ◽  
Dna Plasmid

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

scholarly journals Sepsis Diagnostics: Intensive Care Scoring Systems Superior to MicroRNA Biomarker Testing

Diagnostics ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 701
Author(s):  
Fabian Link ◽  
Knut Krohn ◽  
Anna-Maria Burgdorff ◽  
Annett Christel ◽  
Julia Schumann
Keyword(s):  
Intensive Care ◽  
Scoring Systems ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Medical Problem ◽  
Control Group ◽  
Multiple Parameters ◽  
Sepsis Diagnosis ◽  
Intensive Care Patients ◽  
Healthy Control

Sepsis represents a serious medical problem accounting for numerous deaths of critically ill patients in intensive care units (ICUs). An early, sensitive, and specific diagnosis is considered a key element for improving the outcome of sepsis patients. In addition to classical laboratory markers, ICU scoring systems and serum miRNAs are discussed as potential sepsis biomarkers. In the present prospective observational study, the suitability of miRNAs in sepsis diagnosis was tested based on proper validated and normalized data (i.e., absolute quantification by means of Droplet Digital PCR (ddPCR)) in direct comparison to classical sepsis markers and ICU scores within the same patient cohort. Therefore, blood samples of septic intensive care patients (n = 12) taken at day of admission at ICU were compared to non-septic intensive care patients (n = 12) and a healthy control group (n = 12). Our analysis indicates that all tested biomarkers have only a moderate informative power and do not allow an unequivocal differentiation between septic and non-septic ICU patients. In conclusion, there is no standalone laboratory parameter that enables a reliable diagnosis of sepsis. miRNAs are not superior to classical parameters in this respect. It seems recommendable to measure multiple parameters and scores and to interpret them with regard to the clinical presentation.

Assessment of droplet digital PCR for absolute quantification of genetically engineered OXY235 canola and DP305423 soybean samples

Food Control ◽  
2014 ◽  
Vol 46 ◽  
pp. 470-474 ◽  
Author(s):  
Tigst Demeke ◽  
Tom Gräfenhan ◽  
Michelle Holigroski ◽  
Ursla Fernando ◽  
Janice Bamforth ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Droplet Digital Pcr

scholarly journals Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Water ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3507
Author(s):  
Mark A. Ibekwe ◽  
Shelton E. Murinda ◽  
Stanley Park ◽  
Amarachukwu Obayiuwana ◽  
Marcia A. Murry ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Point Of Care ◽  
Foodborne Pathogen ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
High Rate ◽  
E Coli

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

scholarly journals Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 243 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Masaki Takasu ◽  
Mio Kikuchi ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Genetic Manipulation ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Taqman Probe ◽  
Gene Doping ◽  
Athletic Ability ◽  
Horse Plasma ◽  
Primer Sets

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.

scholarly journals Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of Mycobacterium tuberculosis

2020 ◽  
Vol 8 (5) ◽  
pp. 701 ◽  
Author(s):  
Raphael Nyaruaba ◽  
Jin Xiong ◽  
Caroline Mwaliko ◽  
Nuo Wang ◽  
Belindah J. Kibii ◽  
...  
Keyword(s):  
Target Genes ◽  
Annealing Temperature ◽  
Single Channel ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Sample Concentration ◽  
Specificity And Sensitivity ◽  
Probe Concentration ◽  
Detection And Quantification

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.

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