scholarly journals Digital polymerase chain reaction in an array of microfluidic printed droplets

2019 ◽  
Author(s):  
Yongfan Men ◽  
Jiannan Li ◽  
Tingting Ao ◽  
Zhihao Li ◽  
Bizhu Wu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cost Effective ◽  
Digital Pcr ◽  
Research Field ◽  
Clinical Diagnostics ◽  
Future Market ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
New Perspective ◽  
Digital Polymerase Chain Reaction

AbstractDigital polymerase chain reaction (PCR) is a fast-developed technology, which makes it possible to provide absolute quantitative results. However, this technology has not been widely used in research field or clinical diagnostics. Although digital PCR has been born for two decades, the products on this subject still suffer from either high cost or cumbersome user experience, hence very few labs have the willingness or budget to routinely use such product; On the other hand, the unique sensitivity of dPCR over traditional qPCR shows great potential applications. Here, a cost-effective digital PCR method based on a microfluidic printing system was introduced, trying to overcome those shortcomings. The microfluidic droplet printing technology was utilized in this study to directly generate droplet array containing PCR reaction solution onto the simple glass substrate for the subsequent PCR and imaging, which could be done with any regular flat-panel PCR machine and microscope. The method introduces a new perspective in droplet-based digital PCR in that the droplets generated with this method aligns well in an array without touch with each other, therefore the regular glass and oil could be used without any special surfactant. With simple analysis, the data generated with this method showed reliable quality, which followed the Poisson distribution trend. Compared with other expensive digital PCR methods, this system is more affordable and simpler to integrate, especially for those biological or medical labs which are in need for the digital PCR options but short in budget. Therefore, this method is believed to have the great potential in the future market application.

scholarly journals Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

2020 ◽  
Vol 21 (9) ◽  
pp. 3141 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Giuseppina Tota ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Giorgina Specchia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Residual Disease ◽  
Hematologic Malignancies ◽  
Digital Pcr ◽  
Future Perspectives ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Generation Sequencing ◽  
Minimal Residual

The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology.

scholarly journals Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1022
Author(s):  
Martina Brandolini ◽  
Francesca Taddei ◽  
Maria Michela Marino ◽  
Laura Grumiro ◽  
Agata Scalcione ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Cultures ◽  
Rna Extraction ◽  
Research Field ◽  
Chain Reaction ◽  
Diagnostic Assays ◽  
Qrt Pcr ◽  
Extraction And Purification ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.

Heterogeneous modification of through-hole microwell chips for ultralow cross-contamination digital polymerase chain reaction

The Analyst ◽  
2020 ◽  
Vol 145 (8) ◽  
pp. 3116-3124
Author(s):  
Jinze Li ◽  
Yajun Qiu ◽  
Zhiqi Zhang ◽  
Chuanyu Li ◽  
Shuli Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Digital Pcr ◽  
Cross Contamination ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Through Hole

Heterogeneous modification of through-hole microwell chips to avoid cross-contamination during digital PCR.

scholarly journals Application of droplet digital PCR to detect the pathogens of infectious diseases

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Haiyi Li ◽  
Ruolan Bai ◽  
Zhenyu Zhao ◽  
Lvyan Tao ◽  
Mingbiao Ma ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Infectious Diseases ◽  
Quantitative Polymerase Chain Reaction ◽  
Digital Pcr ◽  
Conventional Polymerase Chain Reaction ◽  
Nucleic Acid Detection ◽  
Chain Reaction ◽  
Molecular Biology Technique ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a molecular biology technique used to multiply certain deoxyribonucleic acid (DNA) fragments. It is a common and indispensable technique that has been applied in many areas, especially in clinical laboratories. The third generation of polymerase chain reaction, droplet digital polymerase chain reaction (ddPCR), is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify DNA. Droplet digital polymerase chain reaction is now widely used in low-abundance nucleic acid detection and is useful in diagnosis of infectious diseases. Here, we summarized the potential advantages of droplet digital polymerase chain reaction in clinical diagnosis of infectious diseases, including viral diseases, bacterial diseases and parasite infections, concluded that ddPCR provides a more sensitive, accurate, and reproducible detection of low-abundance pathogens and may be a better choice than quantitative polymerase chain reaction for clinical applications in the future.

A localized temporary negative pressure assisted microfluidic device for detecting keratin 19 in A549 lung carcinoma cells with digital PCR

2015 ◽  
Vol 7 (5) ◽  
pp. 2006-2011 ◽  
Author(s):  
Qingchang Tian ◽  
Qi Song ◽  
Yanan Xu ◽  
Qiangyuan Zhu ◽  
Bingwen Yu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Digital Pcr ◽  
Carcinoma Cells ◽  
Biological Research ◽  
Chain Reaction ◽  
Lung Carcinoma Cells ◽  
Keratin 19 ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
A549 Lung

Digital polymerase chain reaction (dPCR) has played a major role in biological research, especially by providing an accurate counting of single nucleic acid molecules.

Massive droplets generation for digital PCR via a smart step emulsification chip integrated in a reaction tube

The Analyst ◽  
2021 ◽  
Author(s):  
Shuhao Zhao ◽  
Zengming Zhang ◽  
Fei Hu ◽  
Junjun Wu ◽  
Niancai Peng
Keyword(s):  
Polymerase Chain Reaction ◽  
Large Scale ◽  
Digital Pcr ◽  
Chain Reaction ◽  
Reaction Tube ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Monodisperse Droplets

Step emulsification (SE) devices coupled with parallel generation nozzles is widely used in the production of large-scale monodisperse droplets, especially for droplet based digital polymerase chain reaction (ddPCR) analysis. Although...

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

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.

scholarly journals Development of a Genoserotyping Method for Salmonella Infantis Detection on the Basis of Pangenome Analysis

2020 ◽  
Vol 9 (1) ◽  
pp. 67
Author(s):  
Seung-Min Yang ◽  
Jiwon Baek ◽  
Eiseul Kim ◽  
Hyeon-Be Kim ◽  
Seyoung Ko ◽  
...  
Keyword(s):  
Public Health ◽  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Cost Effective ◽  
The Other ◽  
Gene Marker ◽  
Diagnostic Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Substantial Economic Burden

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

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