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

Mairiang, Dumrong; Songjaeng, Adisak; Hansuealueang, Prachya; Malila, Yuwares; Lertsethtakarn, Paphavee; Silapong, Sasikorn; Poolpanichupatam, Yongyuth; Klungthong, Chonticha; Chin-Inmanu, Kwanrutai; Thiemmeca, Somchai; Tangthawornchaikul, Nattaya; Sriraksa, Kanokwan; Limpitikul, Wannee; Vasanawathana, Sirijitt; Ellison, Damon W.; Malasit, Prida; Suriyaphol, Prapat; Avirutnan, Panisadee

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.