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...

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.

scholarly journals Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1270
Author(s):  
Anna Cutarelli ◽  
Andrea Fulgione ◽  
Pasquale Fraulo ◽  
Francesco Paolo Serpe ◽  
Pasquale Gallo ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Buffalo Milk ◽  
Cow Milk ◽  
Mozzarella Cheese ◽  
Chain Reaction ◽  
New Methods ◽  
Protected Designation Of Origin ◽  
Commission Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

Validation of droplet digital Polymerase Chain Reaction for the detection and absolute quantification of Taenia solium eggs in spiked soil samples

Acta Tropica ◽  
2019 ◽  
Vol 200 ◽  
pp. 105175
Author(s):  
Justine Daudi Maganira ◽  
Beda John Mwang'onde ◽  
Winifrida Kidima ◽  
Chacha John Mwita ◽  
Gamba Nkwengulila ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Taenia Solium ◽  
Absolute Quantification ◽  
Soil Samples ◽  
Chain Reaction ◽  
Spiked Soil ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Application of the Droplet Digital Polymerase Chain Reaction (ddPCR) Platform for Detection and Quantification of Vertebrate Host DNA in Engorged Mosquitoes

2019 ◽  
Vol 56 (4) ◽  
pp. 1150-1153
Author(s):  
L M Rice ◽  
L L Robb ◽  
D A Hartman ◽  
J R Anderson ◽  
R C Kading
Keyword(s):  
Polymerase Chain Reaction ◽  
New Technology ◽  
Field Studies ◽  
Blood Feeding ◽  
Absolute Quantification ◽  
Spatial And Temporal Patterns ◽  
Chain Reaction ◽  
Vector Borne Diseases ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Hematophagous arthropod bloodmeal identification has remained a challenge in the field of vector biology, but these studies are important to understand blood feeding patterns of arthropods, spatial, and temporal patterns in arbovirus transmission cycles, and risk of human and veterinary disease. We investigated the use of an existing vertebrate primer set for use on the droplet digital polymerase chain reaction (ddPCR) platform, to explore the use of this technology in the identification and quantification of vertebrate DNA in mosquito blood meals. Host DNA was detectable 48-h post-engorgement in some mosquitoes by ddPCR, compared with 24-h post-engorgement using traditional PCR. The capability of ddPCR for absolute quantification of template DNA offers unique potential applications of this new technology to field studies on the ecology of vector-borne diseases, but currently with limited scope.

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 Clarity Plus™ digital PCR: A novel platform for absolute quantification of SARS-CoV-2

2021 ◽  
Author(s):  
Shawn Yi Han Tan ◽  
Milton Sheng Yi Kwek ◽  
Huiyu Low ◽  
Yan Ling Joy Pang
Keyword(s):  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Viral Loads ◽  
Nucleic Acid Analysis ◽  
Polymerase Chain ◽  
The Absolute ◽  
Assay Precision ◽  
Digital Polymerase Chain Reaction ◽  
Higher Sensitivity

In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. Considering the growing demand for improved dPCR technology, the Clarity Plus™ dPCR system which features enhanced multiplexing capability and a wider dynamic range for nucleic acid analysis was recently launched. In this study, a dPCR assay optimized for use on Clarity Plus™ was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intra- assay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARS-CoV-2 and can potentially be adopted as a molecular tool in applications such as detecting low viral loads in patients as well as in wastewater surveillance of COVID-19.

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 Detection of fish allergen by droplet digital PCR

2019 ◽  
Vol 7 (4) ◽  
Author(s):  
Cinzia Daga ◽  
Simona Cau ◽  
Maria Giovanna Tilocca ◽  
Barbara Soro ◽  
Aldo Marongiu ◽  
...  
Keyword(s):  
18S Rrna ◽  
Annealing Temperature ◽  
Pcr Primers ◽  
Digital Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
European Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Fish is one of fourteen allergens that must be highlighted on the label within the ingredients list. It should be noted that the European regulation, is very restrictive to allergens with zero tolerance. Therefore it is important to establish sensitive and specific methods for detecting fish allergen. Applicability to detect and quantify fish allergen by droplet digital polymerase chain reaction (ddPCR) has been evaluated in this work. Genomic DNA of three fish species belonging to the most common fish families were analyzed. PCR primers were designed to amplify a 166 bp region of the 18S rRNA gene. Comparative studies were performed to establish the optimal primer and probe concentrations.  Annealing temperature was determined by using thermal gradient. The results have shown good applicability of the optimized 18S rRNA gene-method to detect and quantify small amounts of the target in all samples analyzed. However, validation studies are needed in order to apply ddPCR technology for routine allergens analysis.  

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