Introduction to Digital PCR

Breast cancer is one of the leading causes of cancer-related deaths in women worldwide. Unfortunately, treatments often fail because of the development of drug resistance, the underlying mechanisms of which remain unclear. Circulating tumor DNA (ctDNA) is free DNA released into the blood by necrosis, apoptosis or direct secretion by tumor cells. In contrast to repeated, highly invasive tumor biopsies, ctDNA reflects all molecular alterations of tumors dynamically and captures both spatial and temporal tumor heterogeneity. Highly sensitive technologies, including personalized digital PCR and deep sequencing, make it possible to monitor response to therapies, predict drug resistance and tailor treatment regimens by identifying the genomic alteration profile of ctDNA, thereby achieving precision medicine. This review focuses on the current status of ctDNA biology, the technologies used to detect ctDNA and the potential clinical applications of identifying drug resistance mechanisms by detecting tumor-specific genomic alterations in breast cancer.

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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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Circulating tumor DNA is detectable in canine histiocytic sarcoma, oral malignant melanoma, and multicentric lymphoma

Scientific Reports ◽

10.1038/s41598-020-80332-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anaïs Prouteau ◽

Jérôme Alexandre Denis ◽

Pauline De Fornel ◽

Edouard Cadieu ◽

Thomas Derrien ◽

...

Keyword(s):

Residual Disease ◽

Specific Antigen ◽

Point Mutations ◽

Antigen Receptor ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Histiocytic Sarcoma ◽

Oncology Research ◽

Tumor Dna

AbstractCirculating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.

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Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

Scientific Reports ◽

10.1038/s41598-021-83318-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatsuhiko Hoshino ◽

Ryohei Nakao ◽

Hideyuki Doi ◽

Toshifumi Minamoto

Keyword(s):

Fish Species ◽

High Throughput Sequencing ◽

Correlation Coefficients ◽

Environmental Dna ◽

Digital Pcr ◽

Absolute Quantification ◽

Taqman Probe ◽

Individual Species ◽

Natural Environments ◽

Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

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Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA

Methods ◽

10.1016/j.ymeth.2021.05.006 ◽

2021 ◽

Author(s):

Willem van Snippenberg ◽

David Gleerup ◽

Sofie Rutsaert ◽

Linos Vandekerckhove ◽

Ward de Spiegelaere ◽

...

Keyword(s):

Digital Pcr ◽

Pcr Assays ◽

Hiv 1

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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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