Journal
DIAGNOSTICS
Volume 11, Issue 4, Pages -Publisher
MDPI
DOI: 10.3390/diagnostics11040639
Keywords
dengue virus; droplet digital PCR; virus detection; virus quantification
Categories
Funding
- Faculty of Medicine Siriraj Hospital, Mahidol University [R015834004]
- Thailand Research Fund [RSA 5680049]
- Armed Forces Health Surveillance Division (AFHSD)-Global Emerging Infections Surveillance (GEIS) Branch
- Office of the Higher Education Commission
- Mahidol University under the National Research Universities Initiative
- Siriraj Chalermprakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University
- Research Chair Grant, NSTDA, Thailand
- Research Lecturer Grant, Faculty of Medicine Siriraj Hospital, Mahidol University
- Royal Golden Jubilee Ph.D. Program [PHD/0101/2554]
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RT-ddPCR demonstrates superior sensitivity compared to qRT-PCR in detecting and quantifying DENV, with interlaboratory variations observed in quantification of DENV in clinical samples. This optimized protocol could potentially harmonize DENV quantification results and enhance disease severity correlation studies.
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.
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