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Article

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

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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 Thani 12120, Thailand
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Siriraj Center of Research Excellence in Dengue and Emerging Pathogens, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
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Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
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Graduate Program in Medical Biochemistry and Molecular Biology, Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
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Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathum Thani 12120, Thailand
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Department of Bacterial and Parasitic Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok 10400, Thailand
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Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok 10400, Thailand
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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
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Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
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Pediatric Department, Khon Kaen Hospital, Ministry of Health, Khon Kaen 40000, Thailand
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Pediatric Department, Songkhla Hospital, Ministry of Health, Songkhla 90100, Thailand
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Authors to whom correspondence should be addressed.
These authors contributed equally to this work and are the first authors of this work.
Academic Editor: Nuno Taveira
Diagnostics 2021, 11(4), 639; https://doi.org/10.3390/diagnostics11040639
Received: 4 March 2021 / Revised: 26 March 2021 / Accepted: 26 March 2021 / Published: 1 April 2021
(This article belongs to the Section Diagnostic Microbiology and Infectious Disease)
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 quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity. View Full-Text
Keywords: dengue virus; droplet digital PCR; virus detection; virus quantification dengue virus; droplet digital PCR; virus detection; virus quantification
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MDPI and ACS Style

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. Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens. Diagnostics 2021, 11, 639. https://doi.org/10.3390/diagnostics11040639

AMA Style

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 DW, Malasit P, Suriyaphol P, Avirutnan P. Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens. Diagnostics. 2021; 11(4):639. https://doi.org/10.3390/diagnostics11040639

Chicago/Turabian Style

Mairiang, Dumrong, Adisak Songjaeng, Prachya Hansuealueang, Yuwares Malila, Paphavee Lertsethtakarn, Sasikorn Silapong, Yongyuth Poolpanichupatam, Chonticha Klungthong, Kwanrutai Chin-Inmanu, Somchai Thiemmeca, Nattaya Tangthawornchaikul, Kanokwan Sriraksa, Wannee Limpitikul, Sirijitt Vasanawathana, Damon W. Ellison, Prida Malasit, Prapat Suriyaphol, and Panisadee Avirutnan. 2021. "Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens" Diagnostics 11, no. 4: 639. https://doi.org/10.3390/diagnostics11040639

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