Abachin Eric, Convers Samantha, Falque Stephanie, Esson Raphaël, Mallet Laurent, Nougarede Nolwenn
Analytical R&D Department, Microbiology Characterization R&D Unit, Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France.
Biologicals. 2018 Mar;52:49-54. doi: 10.1016/j.biologicals.2018.01.001. Epub 2018 Feb 3.
Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RT-qPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an R from the linear regression analysis of >0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.
聚合酶链反应（PCR）是一种用于体外扩增核酸的重要分子生物学技术。逆转录定量PCR（RT-qPCR）以及最近的逆转录数字液滴PCR（RT-ddPCR）已被开发用于核酸定量。我们开发了一种用于定量2型减毒登革病毒核酸的RT-ddPCR检测方法，并将其与常规RT-qPCR检测方法进行了比较。常规RT-qPCR检测方法靶向NS5基因，而RT-ddPCR检测方法的条件优化则选择了E基因。使用单独的2型减毒登革病毒以及在其他三种登革病毒血清型存在的情况下，证明了该检测方法的特异性。对25份2型减毒登革病毒样本进行的两种检测结果具有可比性，线性回归分析的R值>0.98。RT-ddPCR检测方法的一个主要优点是它无需标准曲线即可进行核酸定量。在疫苗生产过程中，RT-ddPCR可用于登革疫苗病毒核酸的绝对定量。
