Dumrong MairiangAdisak SongjaengPrachya HansuealueangYuwares MalilaPaphavee LertsethtakarnSasikorn SilapongYongyuth PoolpanichupatamChonticha KlungthongKwanrutai Chin-InmanuSomchai ThiemmecaNattaya TangthawornchaikulKanokwan SriraksaWannee LimpitikulSirijitt VasanawathanaDamon W. EllisonPrida MalasitPrapat SuriyapholPanisadee AvirutnanSiriraj HospitalSongkhla HospitalArmed Forces Research Institute of Medical Sciences, ThailandKhon Kaen Regional HospitalThailand National Center for Genetic Engineering and Biotechnology2022-08-042022-08-042021-04-01Diagnostics. Vol.11, No.4 (2021)207544182-s2.0-85108986648https://repository.li.mahidol.ac.th/handle/20.500.14594/76216Detection 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.3390/diagnostics11040639