Data Sheet 1_Development and validation of a duplex droplet digital PCR assay for the simultaneous detection of cytomegalovirus and Epstein-Barr virus in plasma.docx

BackgroundHuman cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are globally prevalent herpesviruses. While typically self-limiting in immunocompetent individuals, infections can cause severe consequences even in this population. In immunocompromised groups, such as transplant recipients and HIV-infected individuals, viral reactivation or coinfection frequently triggers graft rejection, multi-organ invasion, and malignancies, often exhibiting synergistic pathogenicity. Current serological assays are limited by “window periods” and delayed immune responses, while traditional quantitative PCR (qPCR) relies on standard curves for quantification. Consequently, there is an urgent need for precise, interference-resistant methods. This study aimed to develop and validate a duplex droplet digital PCR (ddPCR) assay for the simultaneous, absolute quantification of CMV and EBV in plasma.
MethodsBased on the TD-1 platform, a single-tube duplex detection system targeting conserved viral regions was optimized to minimize the “rain effect” and maximize signal-to-noise ratios. Leveraging the absolute quantification capability of ddPCR without standard curves, we compared its performance, including dynamic range and limit of detection (LOD), against a homologous qPCR assay. Clinical validation was conducted using 117 plasma samples from suspected cases, utilizing a commercial qPCR kit as the reference standard. Additionally, tolerance to endogenous interfering substances was assessed.
ResultsThe optimized duplex ddPCR demonstrated high analytical sensitivity, with LODs for CMV and EBV at 7.9 and 6.5 copies/reaction, respectively, which were approximately 6- to 7-fold lower than homologous qPCR (53.4 and 45.6 copies/reaction).No competitive inhibition was observed at low concentrations. In clinical validation, the assay achieved 100% sensitivity and specificity compared to the reference kit, with high quantitative correlation (R2 = 0.80–0.87). Notably, ddPCR detected four weak positive samples (1 CMV, 3 EBV) missed by homologous qPCR. Furthermore, the method maintained accurate quantification in plasma containing hyperlipidemia or hyperbilirubinemia.
ConclusionThis study successfully established a duplex ddPCR assay characterized by high sensitivity, specificity, and robust interference resistance. By enabling precise absolute quantification, it serves as a powerful complement to existing technologies for the early diagnosis and monitoring of CMV and EBV active infections.