Droplet digital PCR applications in the tuberculosis world

Nyaruaba, Raphael; Mwaliko, Caroline; Kering, Kelvin; Wei, Hongping

doi:10.1016/j.tube.2019.07.001

Cited by 65 publications

(69 citation statements)

References 49 publications

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“…Experimentally linking SNPs over distances exceeding those that can be amplified by long-range PCR (up to approximately 20 kb) remains a challenge. Although technologies such as 10X Genomics Linked-Reads (discussed in more detail below) are establishing themselves as powerful tools, cost is a major barrier for their use in routine clinical and research applications and the vendor has recently announced that support for the platform will end in July, 2020. ddPCR is increasingly utilized for DNA and RNA quantification, detection of tumor-derived DNA and pathogen detection to name a few (Li et al, 2018;Liao et al, 2019;Nyaruaba et al, 2019;O'Hara et al, 2019;Oscorbin et al, 2019;Valpione and Campana, 2019), but can also be employed for linkage analysis (Roberts et al, 2014;Karlin-Neumann and Bizouarn, 2018;Lunenburg et al, 2018;Regan and Karlin-Neumann, 2018;Tsujimoto et al, 2018). Since assays can easily be developed by the user and cost-effectively performed, DropPhase2D6 was established to experimentally determine linkage between the "enhancer" SNP and known CYP2D6 haplotypes.…”

Section: Dropphase2d6mentioning

confidence: 99%

Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

et al. 2020

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Background: The CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation, or structural rearrangements. It was proposed that rs5758550, located 116 kb downstream of the CYP2D6 gene locus, increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, also referred to as the "enhancer" single-nucleotide polymorphism (SNP), to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the "enhancer" SNP to interindividual variability in CYP2D6 activity. Methods: A large ethnically mixed population sample (n=3,162) was computationally phased to determine linkage between the "enhancer" SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept. Results: Phasing predicted that the "enhancer" SNP can occur on numerous CYP2D6 haplotypes including CYP2D6*1, *2, *5, and *41 and suggested that linkage is incomplete, i.e., a portion of these alleles do not have the "enhancer" SNP. Phasing also revealed differences among the European and African ancestry data sets regarding the proportion of alleles with and without the "enhancer" SNP. DropPhase2D6 was utilized to confirm or refute the predicted "enhancer" SNP location for individual samples, e.g., of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on the *1 allele of one sample. Our findings highlight that the location of the "enhancer" SNP must not be

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Section: Dropphase2d6mentioning

confidence: 99%

Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

et al. 2020

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“…Since systemic MCMV infection of mice early in life resulted in MCMV latency in the choroid/RPE, we hypothesized that HCMV might spread to and become latent in the choroid/RPE of human eyes. To test this hypothesis, the anterior segment (cornea, ciliary body, and iris), posterior eye cup (choroid/RPE), and retina (neural retina) were dissected from 48 fresh eyes of 24 human cadavers obtained from Georgia Eye Bank, Inc. DNA was extracted and analyzed for the presence of HCMV DNA by ddPCR, a technology emulsifying an oil-based PCR reaction into thousands of droplets, each of which then acts as a PCR micro-reaction and increases the chances of rare target sequences being detected [63], as evidenced by its use in the detection [64][65][66] including latent HCMV infections [67,68]. As shown in supplementary material, Table S2, HCMV DNA was detected in four posterior eye cups (choroid/RPE) of four individual cadavers.…”

Section: Hcmv Dna Is Present In Human Ocular Tissuementioning

confidence: 99%

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization

Liu

Zhang

et al. 2020

The Journal of Pathology

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Age‐related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50–80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro‐angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)‐overexpressing VEGF‐Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF‐overexpressingVEGF‐Ahyper and wild‐type(WT) 129 mice. By 6 months post‐infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF‐Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF‐Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV‐infected VEGF‐Ahyper mice compared to eyes of uninfected VEGF‐Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL‐6, were significantly higher in eyes of MCMV‐infected VEGF‐Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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“…Poisson statistics [8,9], which works by separating a sample into thousands to millions of droplets and then partitioning them to be read as either positive or negative depending on uorescence amplitude [10][11][12][13]. These vast and highly consistent oil droplets substantially improve the detection dynamic range and accuracy of ddPCR [14].…”

Section: Droplet Digital Pcr (Ddpcr) Is a Third Generation Pcr Based mentioning

confidence: 99%

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Zhang

Dai

Wang

et al. 2020

Preprint

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Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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Droplet digital PCR applications in the tuberculosis world

Cited by 65 publications

References 49 publications

Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

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