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.

A rapid nucleic acid concentration measurement system with large-field-of-view for droplet digital PCR microfluidic chip

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Jinrong Shen ◽  
Jihong Zheng ◽  
Zhenqing Li ◽  
Yourong Liu ◽  
Fengxiang Jing ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Field Of View ◽  
Large Field ◽  
Chain Reaction ◽  
Effective Technique ◽  
Nucleic Acid Concentration ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction(ddPCR) is an effective technique for the absolute quantification of target mucleic acid unparalleled sensitivity. However, current commerical ddPCR device for the detection of the gene...

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 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 Digital PCR as an Emerging Tool for Monitoring of Microbial Biodegradation

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 706 ◽  
Author(s):  
Yiqi Cao ◽  
Miao Yu ◽  
Guihua Dong ◽  
Bing Chen ◽  
Baiyu Zhang
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Functional Genes ◽  
Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Quantification Method ◽  
Monitoring Technique ◽  
Microbial Biodegradation ◽  
Polymerase Chain ◽  
Pcr Techniques

Biodegradation of contaminants is extremely complicated due to unpredictable microbial behaviors. Monitoring of microbial biodegradation drives us to determine (1) the amounts of specific degrading microbes, (2) the abundance, and (3) expression level of relevant functional genes. To this endeavor, the cultivation independent polymerase chain reaction (PCR)-based monitoring technique develops from endpoint PCR, real-time quantitative PCR, and then into novel digital PCR. In this review, we introduce these three categories of PCR techniques and summarize the timely applications of digital PCR and its superiorities than qPCR for biodegradation monitoring. Digital PCR technique, emerging as the most accurately absolute quantification method, can serve as the most promising and robust tool for monitoring of microbial biodegradation.

scholarly journals Multiple cyanotoxin congeners produced by sub-dominant cyanobacterial taxa in riverine cyanobacterial and algal mats

2019 ◽  
Author(s):  
Laura T. Kelly ◽  
Keith Bouma-Gregson ◽  
Jonathan Puddick ◽  
Rich Fadness ◽  
Ken G. Ryan ◽  
...  
Keyword(s):  
Green Alga ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Wilcoxon Test ◽  
Cladophora Glomerata ◽  
Chain Reaction ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Polymerase Chain ◽  
Russian River

AbstractBenthic cyanobacterial proliferations in rivers are have been reported with increasing frequency worldwide. In the Eel and Russian rivers of California, more than a dozen dog deaths have been attributed to cyanotoxin toxicosis since 2000. Periphyton proliferations in these rivers comprise multiple cyanobacterial taxa capable of cyanotoxin production, hence there is uncertainty regarding which taxa are producing toxins. In this study, periphyton samples dominated by the cyanobacterial genera Anabaena spp. and Microcoleus spp. and the green alga Cladophora glomerata were collected from four sites in the Eel River catchment and one site in the Russian River. Samples were analysed for potential cyanotoxin producers using polymerase chain reaction (PCR) in concert with Sanger sequencing. Cyanotoxin concentrations were measured using liquid chromatography tandem-mass spectrometry, and anatoxin quota determined using droplet digital PCR. Sequencing indicated Microcoleus sp. and Nodularia sp. were the putative producers of anatoxins and nodularins, respectively, regardless of the dominant taxa in the mat. Anatoxin concentrations in the mat samples varied from 0.1 to 18.6 μg g−1 and were significantly different among sites (p < 0.01, Wilcoxon test); however, anatoxin quotas were less variable (< 5-fold). Dihydroanatoxin-a was generally the most abundant variant in samples comprising 38% to 71% of the total anatoxins measured. Mats dominated by the green alga C. glomerata contained both anatoxins and nodularin-R at concentrations similar to those of cyanobacteria-dominated mats. This highlights that even when cyanobacteria are not the dominant taxa in periphyton, these mats may still pose a serious health risk and indicates that more widespread monitoring of all mats in a river are necessary.

scholarly journals Quantitative real-time polymerase chain reaction (PCR) and droplet digital PCR duplex assays for detectingZostera marinaDNA in coastal sediments

2018 ◽  
Vol 16 (4) ◽  
pp. 253-264 ◽  
Author(s):  
Masami Hamaguchi ◽  
Hiromori Shimabukuro ◽  
Masakazu Hori ◽  
Goro Yoshida ◽  
Tomoko Terada ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Coastal Sediments ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

Insects ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1034
Author(s):  
Darja Kušar ◽  
Bojan Papić ◽  
Urška Zajc ◽  
Irena Zdovc ◽  
Majda Golob ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Germination Rate ◽  
Single Copy ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Qpcr Assay ◽  
Paenibacillus Larvae ◽  
Reliable Quantification ◽  
Detection And Quantification

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

scholarly journals Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR?

2019 ◽  
Vol 20 (9) ◽  
pp. 2249 ◽  
Author(s):  
Daniela Cilloni ◽  
Jessica Petiti ◽  
Valentina Rosso ◽  
Giacomo Andreani ◽  
Matteo Dragani ◽  
...  
Keyword(s):  
Residual Disease ◽  
Myeloproliferative Disorders ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Acute Leukemias ◽  
New Techniques ◽  
Standard Curve ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Digital Polymerase Chain Reaction

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.

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