scholarly journals Rapid and Reliable Detection of Echinococcus multilocularis from Faeces Using Droplet Digital PCR

2020 ◽  
Author(s):  
Fabian Bagó ◽  
Franz Hoelzl ◽  
Felix Knauer ◽  
Anna Kübber-Heiss ◽  
Steve Smith
Keyword(s):  
High Throughput ◽  
Echinococcus Multilocularis ◽  
Current Method ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Diagnostic Methods ◽  
Credibility Interval ◽  
Red Foxes ◽  
Wild Canids ◽  
Magnetic Capture

Abstract Purpose Alveolar echinococcosis is a severe helminthic disease in humans caused by larvae of the fox tapeworm Echinococcus multilocularis. Austria is considered an endemic area with hotspots having up to 45% prevalence (Bagó et al. in Proceedings of the Zoo and Wildlife Health Conference 2019, Berlin, p. 91, 2019). At our facility, we have registered a notifiable increase of animals submitted for the diagnosis of E. multilocularis since 2016. Therefore, we investigated high throughput diagnostic methods to provide rapid and reliable results in comparison with our current method. Methods We have developed and compared a novel method of detection using droplet digital PCR (ddPCR) combined with previous target specific extraction according to Maas et al. (Vet Parasitol 230:20–24, 2016), with our current macroscopic method “Shaking in a Vessel Technique” (SVT) by Duscher et al. (Parasitol Res 95(1):40–42, 2005). We investigated 77 wild canids (72 red foxes, 5 golden jackals) using both methods. The data were analyzed using a non-Bayesian approach, applying bootstrapping to create confidentiality intervals. Results Sensitivity for droplet digital PCR was 90.51% with the 95% credibility interval ranging from 82.50 to 96.92%, whereas mean sensitivity for SVT was 92.04% with a 95% credibility interval ranging from 84.75% to 98.36%. Additionally, a non-linear regression similar to R2 could be pointed out between the counted worms and the results gathered from ddPCR. Conclusion Magnetic capture extraction followed by ddPCR shows strong potential as a high throughput method for diagnosing E. multilocularis prevalence in diverse canid populations as well as infection intensities of individual animals, giving valuable epidemiological insights of the distribution amongst wild canids as an alternative to conventional qPCR or macroscopic methods.

scholarly journals Direct Comparison of SARS-CoV-2 Analytical Limits of Detection across Seven Molecular Assays

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Becky Fung ◽  
Allan Gopez ◽  
Venice Servellita ◽  
Shaun Arevalo ◽  
Coral Ho ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Point Of Care ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Limits Of Detection ◽  
Molecular Assays ◽  
Performance Metric ◽  
Roche Cobas

ABSTRACT Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.

scholarly journals High-Throughput Detection of Multiple miRNAs and Methylated DNA by Droplet Digital PCR

2021 ◽  
Vol 11 (5) ◽  
pp. 359
Author(s):  
Ning Li ◽  
Pushpa Dhilipkannah ◽  
Feng Jiang
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Early Detection ◽  
Cancer Patients ◽  
High Throughput ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Lung Cancer Patients ◽  
Molecular Aberrations ◽  
High Throughput Assay

Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a synergistic effect on the diagnosis of malignancies. Herein, we develop a high-throughput assay for detecting multiple miRNAs and DNA methylation using droplet digital PCR (ddPCR) coupled with a 96-microwell plate. The microplate-based ddPCR could absolutely and reproducibly quantify 15 miRNAs and 14 DNA methylation sites with a high sensitivity (one copy/µL and 0.1%, respectively). Analyzing sputum and plasma of 40 lung cancer patients and 36 cancer-free smokers by this approach identified an integrated biomarker panel consisting of two sputum miRNAs (miRs-31-5p and 210-3p), one sputum DNA methylation (RASSF1A), and two plasma miRNAs (miR-21-5p and 126) for the diagnosis of lung cancer with higher sensitivity and specificity compared with a single type of biomarker. The diagnostic value of the integrated biomarker panel for the early detection of lung cancer was confirmed in a different cohort of 36 lung cancer patients and 39 cancer-free smokers. The high-throughput assay for quantification of multiple molecular aberrations across sputum and plasma could improve the early detection of lung cancer.

Productive screening of single aptamers with ddPCR

The Analyst ◽  
2020 ◽  
Vol 145 (12) ◽  
pp. 4130-4137
Author(s):  
Jinyu Chen ◽  
Jinjun Wang ◽  
Zhaofeng Luo ◽  
Xiaona Fang ◽  
Lei He ◽  
...  
Keyword(s):  
High Throughput ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Discovery Method

We have developed an aptamer discovery method that combines a capillary-based integrated droplet digital PCR system with cytometer sorting of beads in a high-throughput way.

scholarly journals Echinococcus species from red foxes, corsac foxes, and wolves in Mongolia

Parasitology ◽  
2013 ◽  
Vol 140 (13) ◽  
pp. 1648-1654 ◽  
Author(s):  
AKIRA ITO ◽  
GANTIGMAA CHULUUNBAATAR ◽  
TETSUYA YANAGIDA ◽  
ANU DAVAASUREN ◽  
BATTULGA SUMIYA ◽  
...  
Keyword(s):  
Life Cycle ◽  
Host Specificity ◽  
Echinococcus Multilocularis ◽  
Red Foxes ◽  
Echinococcus Canadensis ◽  
Wild Canids ◽  
Small Intestines

SUMMARYThe small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.

scholarly journals High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

2011 ◽  
Vol 83 (22) ◽  
pp. 8604-8610 ◽  
Author(s):  
Benjamin J. Hindson ◽  
Kevin D. Ness ◽  
Donald A. Masquelier ◽  
Phillip Belgrader ◽  
Nicholas J. Heredia ◽  
...  
Keyword(s):  
High Throughput ◽  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Dna Copy Number ◽  
Absolute Quantitation

scholarly journals Rapid high-resolution measurement of DNA replication timing by droplet digital PCR

2017 ◽  
Author(s):  
Dzmitry G. Batrakou ◽  
Emma D. Heron ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Replication Timing ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Human Cells ◽  
Genome Replication ◽  
Multiple Loci ◽  
Dna Replication Timing

ABSTRACTGenomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.

Droplet digital PCR as a sensitive tool to assess exposure pressure from Echinococcus multilocularis in intermediate hosts

Acta Tropica ◽  
2021 ◽  
pp. 106078
Author(s):  
Alessandro Massolo ◽  
Amanda Gerber ◽  
Gerald Umhang ◽  
Colin Nicholas ◽  
Stefano Liccioli ◽  
...  
Keyword(s):  
Echinococcus Multilocularis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Intermediate Hosts ◽  
Sensitive Tool

High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70

2014 ◽  
Vol 207 ◽  
pp. 169-177 ◽  
Author(s):  
Motokazu Mukaide ◽  
Masaya Sugiyama ◽  
Masaaki Korenaga ◽  
Kazumoto Murata ◽  
Tatsuya Kanto ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
High Throughput ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Next Generation ◽  
Pcr Assay

Real-time PCR and droplet digital PCR are accurate and reliable methods to quantify Pseudomonas syringae pv. actinidiae biovar 3 in kiwifruit infected plantlets

Plant Disease ◽  
2020 ◽  
Author(s):  
Kelvina Barrett-Manako ◽  
Mark Thomas Andersen ◽  
Marcela Martínez-Sánchez ◽  
Heather Jenkins ◽  
Shannon Hunter ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
High Throughput ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Alternative Methods ◽  
Economic Losses ◽  
Counting Method ◽  
Method Performance ◽  
Colony Counting ◽  
Qpcr Method

Pseudomonas syringae pv. actinidiae (Psa) is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence and rate of infection in kiwifruit cultivars is important in applying effective measures of disease control. Psa load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work we used three alternative Psa quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative qPCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared to the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of Psa3 inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 0.955) and flexible allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods’ accuracy. The qPCR method was efficient and allowed for high-throughput processing and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification and useful for research studies e.g. disease phenotyping and host-pathogen interactions.

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