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 Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 457 ◽  
Author(s):  
Teruaki Tozaki ◽  
Aoi Ohnuma ◽  
Mio Kikuchi ◽  
Taichiro Ishige ◽  
Hironaga Kakoi ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Limit Of Detection ◽  
Doping Control ◽  
Taqman Probe ◽  
Specific Detection ◽  
Gene Doping ◽  
Athletic Ability ◽  
Horse Plasma ◽  
Fluidic Circuits ◽  
Primer Sets

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

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.

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 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.

scholarly journals Absolute quantification by droplet digital PCR versus analog real-time PCR

2013 ◽  
Vol 10 (10) ◽  
pp. 1003-1005 ◽  
Author(s):  
Christopher M Hindson ◽  
John R Chevillet ◽  
Hilary A Briggs ◽  
Emily N Gallichotte ◽  
Ingrid K Ruf ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr

Absolute quantification of intrahepatic HBV covalently-closed-circular DNA by droplet digital PCR

2017 ◽  
Vol 49 (1) ◽  
pp. e36
Author(s):  
G.P. Caviglia ◽  
M.L. Abate ◽  
A. Olivero ◽  
C. Rosso ◽  
A. Ciancio ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Circular Dna ◽  
Covalently Closed Circular Dna ◽  
Intrahepatic Hbv

scholarly journals Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events

2020 ◽  
Author(s):  
Tigst Demeke ◽  
Monika Eng ◽  
Michelle Holigroski ◽  
Sung-Jong Lee
Keyword(s):  
Zero Tolerance ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Genetically Engineered ◽  
Droplet Digital Pcr ◽  
Chain Reaction ◽  
Low Level ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Detection And Quantification

Abstract Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples.

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