During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.

在疫苗生产过程中，目前使用单独的血清型特异性 RT-qPCR 检测对来自嵌合黄热病登革热 (CYD) 疫苗病毒（CYD1、CYD2、CYD3 和 CYD4）的 RNA 进行量化。在这里，我们描述了一项概念验证研究的结果，该研究开发了多重逆转录酶液滴数字 PCR (RT-ddPCR) 检测，用于同时定量所有四种病毒的 RNA。使用常规 RT-qPCR 中使用的血清型特异性 PCR 系统（正向和反向引物和 FAM（荧光发色团 6-羧基荧光素）和 YY（亚基马黄）标记的探针）开发了血清型特异性单链 RT-ddPCR。PCR 系统具有特异性，并给出了与 RT-qPCR 分析相似的定量结果。线性回归分析用于选择相对探针浓度，以获得多重 RT-ddPCR 检测中二维簇图中每个目标 RNA 的不同簇。我们展示了每个 CYD RNA 的簇的位置与模型中预测的一样，并且被很好地分开。对于含有 7、8 或 9 Log10 Geq/mL 的三份样品，多重 RT-ddPCR 得到的定量结果与通过血清型特异性 RT-qPCR 检测获得的结果相似。总之，这些结果表明，可以在单次检测中使用多重 RT-ddPCR 检测对来自四种 CYD 血清型的 RNA 进行定量。对于含有 7、8 或 9 Log10 Geq/mL 的三份样品，多重 RT-ddPCR 得到的定量结果与通过血清型特异性 RT-qPCR 检测获得的结果相似。总之，这些结果表明，可以在单次检测中使用多重 RT-ddPCR 检测对来自四种 CYD 血清型的 RNA 进行定量。对于含有 7、8 或 9 Log10 Geq/mL 的三份样品，多重 RT-ddPCR 得到的定量结果与通过血清型特异性 RT-qPCR 检测获得的结果相似。总之，这些结果表明，可以在单次检测中使用多重 RT-ddPCR 检测对来自四种 CYD 血清型的 RNA 进行定量。