Droplet Digital PCR: Resolving difficult challenges in nucleic acid detection and quantitation

2016 ◽  
Author(s):  
Francisco Bizouarn
Keyword(s):  
Nucleic Acid ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Nucleic Acid Detection

scholarly journals Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing

2021 ◽  
Author(s):  
Sang-Soo Lee ◽  
Seil Kim ◽  
Hee Min Yoo ◽  
Da-Hye Lee ◽  
Young-Kyung Bae
Keyword(s):  
Nucleic Acid ◽  
Copy Number ◽  
Target Gene ◽  
Target Genes ◽  
Digital Pcr ◽  
Number Concentration ◽  
Droplet Digital Pcr ◽  
Molecular Testing ◽  
Testing Laboratories ◽  
Viral Target

AbstractNucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

scholarly journals Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yuri Uchiyama ◽  
Mitsuko Nakashima ◽  
Satoshi Watanabe ◽  
Masakazu Miyajima ◽  
Masataka Taguri ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Somatic Mutation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Leukocytes ◽  
Target Nucleic Acid ◽  
Weber Syndrome ◽  
Sturge Weber Syndrome ◽  
Blood Leukocyte

Abstract Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA–ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge–Weber syndrome (SWS) using ddPCR and PNA–ddPCR. Importantly, among three patients previously found to be mutation negative by next–generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.

scholarly journals Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid

Biologicals ◽  
2018 ◽  
Vol 52 ◽  
pp. 49-54 ◽  
Author(s):  
Eric Abachin ◽  
Samantha Convers ◽  
Stephanie Falque ◽  
Raphaël Esson ◽  
Laurent Mallet ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Digital Pcr ◽  
Droplet Digital Pcr

A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies

The Analyst ◽  
2019 ◽  
Vol 144 (7) ◽  
pp. 2239-2247 ◽  
Author(s):  
Chianru Tan ◽  
Xihua Chen ◽  
Fang Wang ◽  
Dong Wang ◽  
Zongfu Cao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Prenatal Testing ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Nucleic Acid Probes ◽  
Non Invasive ◽  
Fetal Aneuploidies

Using universal locked nucleic acid probes, a high multiplexing ddPCR-based NIPT was developed to reliably identify fetal aneuploidies.

scholarly journals Accurate bulk quantitation of droplet digital PCR

2021 ◽  
Author(s):  
Chen Sun ◽  
Leqian Liu ◽  
Harish N. Vasudevan ◽  
Kai-Chun Chang ◽  
Adam R. Abate
Keyword(s):  
Nucleic Acid ◽  
High Volume ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Settings ◽  
Sample Processing ◽  
Bulk Analysis ◽  
Processing Ability ◽  
Novel Method ◽  
Bulk Processing

AbstractDroplet digital PCR provides superior accuracy in nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource or clinical settings. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.

scholarly journals Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

2019 ◽  
Author(s):  
Samuel Long ◽  
Brian Berkemeire
Keyword(s):  
Rhesus Macaques ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Viral Detection ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Key Aspects ◽  
Hiv 1

AbstractHighly sensitive detection of HIV-1 nucleic acids is of critical importance for evaluating treatment interventions superimposed on combination antiretroviral therapy (cART) in HIV-1 infected individuals. SIV infection of rhesus macaques models many key aspects of human HIV-1 infection and plays a key role in evaluation of approaches for prevention, treatment and attempted eradication of HIV infection. Here we describe ultrasensitive droplet digital PCR (ddPCR) DNA and RT-ddPCR RNA assays for detecting simian immunodeficiency virus (SIV) on the Raindance ddPCR platform. We demonstrate that RainDance ddPCR can tolerate significantly higher cell DNA input without inhibition on a per reaction basis, exceeding the Bio-Rad ddPCR per-reaction input limit by about 18-fold, effectively increasing viral detection sensitivity by allowing a large quantity of sample to be analyzed in each reaction. In addition, the combination of a high processivity RT enzyme and RainDance ddPCR could overcome inhibition in severely inhibited viral RNA samples. These assays offer valuable tools for assessing low level viral production/replication and strategies for targeting residual virus in the setting of cART suppression of viral replication. The methodologies presented here can be adapted for a broad range of applications where highly sensitive nucleic acid detection is required.

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