ddpcRquant: threshold determination for single channel droplet digital PCR experiments

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.

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

Endocrine Abstracts ◽

10.1530/endoabs.42.il5 ◽

2016 ◽

Author(s):

Francisco Bizouarn

Keyword(s):

Nucleic Acid ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Nucleic Acid Detection

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Detection of lncRNAs in thyroid nodule as new tool for tumor diagnosis: analysis by Droplet Digital PCR in Fine Needle Aspiration biopsy

Endocrine Abstracts ◽

10.1530/endoabs.56.gp241 ◽

2018 ◽

Author(s):

Simona Nanni ◽

Pietro Locantore ◽

Lorenza Bacci ◽

Aiello Aurora ◽

Guido Fadda ◽

...

Keyword(s):

Thyroid Nodule ◽

Fine Needle Aspiration ◽

Fine Needle Aspiration Biopsy ◽

Digital Pcr ◽

Needle Aspiration ◽

Droplet Digital Pcr ◽

Tumor Diagnosis ◽

Aspiration Biopsy ◽

Fine Needle ◽

Needle Aspiration Biopsy

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Droplet digital PCR quantification of norovirus and adenovirus in decentralized wastewater and graywater collections: Implications for onsite reuse

Water Research ◽

10.1016/j.watres.2019.115213 ◽

2020 ◽

Vol 169 ◽

pp. 115213 ◽

Cited By ~ 8

Author(s):

Michael A. Jahne ◽

Nichole E. Brinkman ◽

Scott P. Keely ◽

Brian D. Zimmerman ◽

Emily A. Wheaton ◽

...

Keyword(s):

Digital Pcr ◽

Droplet Digital Pcr

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Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

European Food Research and Technology ◽

10.1007/s00217-021-03786-y ◽

2021 ◽

Author(s):

Christian Schulze ◽

Anne-Catrin Geuthner ◽

Dietrich Mäde

Keyword(s):

Durum Wheat ◽

Real Time ◽

Bread Wheat ◽

Common Wheat ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Food Fraud ◽

Single Genome ◽

Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

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Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib

Biomedicines ◽

10.3390/biomedicines9070813 ◽

2021 ◽

Vol 9 (7) ◽

pp. 813

Author(s):

Michele Manganelli ◽

Ilaria Grossi ◽

Manuela Ferracin ◽

Paola Guerriero ◽

Massimo Negrini ◽

...

Keyword(s):

Digital Pcr ◽

Droplet Digital Pcr ◽

Healthy Individuals ◽

Roc Curve Analysis ◽

Multikinase Inhibitor ◽

Sorafenib Treatment ◽

The Third ◽

Circulating Levels ◽

Systemic Drug

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

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