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Application of one-step reverse transcription droplet digital pcr for dengue virus detection and quantification in clinical specimens
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Metadata
Document Title
Application of one-step reverse transcription droplet digital pcr for dengue virus detection and quantification in clinical specimens
Author
Mairiang D.,Songjaeng A.,Hansuealueang P.,Malila Y.,Lertsethtakarn P.,Silapong S.,Poolpanichupatam Y.,Klungthong C.,Chin-Inmanu K.,Thiemmeca S.,Tangthawornchaikul N.,Sriraksa K.,Limpitikul W.,Vasanawathana S.,Ellison D.W.,Malasit P.,Suriyaphol P.,Avirutnan P.
Name from Authors Collection
Scopus Author ID
6507262736
Affiliations
Molecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathum Thani12120, Thailand; Siriraj Center of Research Excellence in Dengue and Emerging Pathogens, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathum Thani12120, Thailand; Department of Bacterial and Parasitic Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, 10400, Thailand; Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, 10400, Thailand; Division of Bioinformatics and Data Management for Research, Research Group and Research Network Division, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand; Pediatric Department, Khon Kaen Hospital, Ministry of Health, Khon Kaen, 40000, Thailand; Pediatric Department, Songkhla Hospital, Ministry of Health, Songkhla, 90100, Thailand
Type
Article
Source Title
Diagnostics
ISSN
20754418
Year
2021
Volume
11
Issue
4
Open Access
All Open Access, Gold, Green
Publisher
MDPI
DOI
10.3390/diagnostics11040639
Abstract
Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantifi cation results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity. © 2021 by the authors.
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
License
CC BY
Rights
Author
Publication Source
Scopus