Mantel N, Aguirre M, Gulia S, Girerd-Chambaz Y, Colombani S, Moste C, Barban V
Microbiology Research Department, Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy l'Etoile, France.
J Virol Methods. 2008 Jul;151(1):40-6. doi: 10.1016/j.jviromet.2008.03.026. Epub 2008 May 22.
Yellow fever-dengue chimeras (CYDs) are being developed currently as live tetravalent dengue vaccine candidates. Specific quantitative assays are needed to evaluate the viral load of each serotype in vaccine batches and biological samples. A quantitative real-time RT-PCR (qRT-PCR) system was developed comprising five one-step qRT-PCRs targeting the E/NS1 junction of each chimera, or the NS5 gene in the yellow fever backbone. Each assay was standardized using in vitro transcribed RNA qualified according to its size and purity, and precisely quantified. A non RNA-extracted virus sample was introduced as external quality control (EQC), as well as 2 extraction controls consisting of 2 doses, 40 and 4,000 GEQ (genomic equivalents), of this EQC extracted in parallel to the samples. Between 6 and 10 GEQ/reaction were reproducibly measured with all assays and similar titers were obtained with the two methods when chimeric virus samples were quantified with the E/NS1- or the NS5-specific assays. Reproducibility of RNA extraction was ensured by automation of the process (yield>or=50%), and infectious virus was isolated in >or=80% of PCR-positive sera from immune monkeys.
黄热病 - 登革热嵌合体（CYD）目前正作为四价登革热活疫苗候选物进行研发。需要特定的定量检测方法来评估疫苗批次和生物样品中各血清型的病毒载量。我们开发了一种定量实时逆转录聚合酶链反应（qRT-PCR）系统，该系统包含五个一步法qRT-PCR，分别靶向每种嵌合体的E/NS1连接处或黄热病毒骨架中的NS5基因。每种检测方法都使用根据其大小和纯度鉴定合格的体外转录RNA进行标准化，并进行精确量化。引入一个未提取RNA的病毒样品作为外部质量控制（EQC），以及2个提取对照，它们由与样品平行提取的2剂、分别为40和4000基因组当量（GEQ）的该EQC组成。所有检测方法均可重复性地检测到每个反应6至10个GEQ，当用E/NS1特异性或NS5特异性检测方法对嵌合病毒样品进行定量时，两种方法获得的滴度相似。通过该过程的自动化确保了RNA提取的可重复性（产量≥50%），并且在≥80%的来自免疫猴子的PCR阳性血清中分离出了感染性病毒。
