使用定量实时聚合酶链反应估计活病毒疫苗中感染性麻疹病毒的数量。

Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR.

作者信息

Schalk Johanna A C, van den Elzen Christa, Ovelgönne Hans, Baas Chantal, Jongen Peter M J M

机构信息

National Institute of Public Health and the Environment (RIVM), Centre for Biological Medicines and Medical Technology, Antonie van Leeuwenhoeklaan 9, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.

出版信息

J Virol Methods. 2004 May;117(2):179-87. doi: 10.1016/j.jviromet.2004.01.009.

DOI:10.1016/j.jviromet.2004.01.009

PMID:15041215

Abstract

The potency of live attenuated virus vaccines is determined by counting or titrating viable viruses in cell cultures. These classical potency tests have the drawback that they are time consuming and laborious and show a high laboratory-to-laboratory variation. In the present study we describe the development and validation of a fast method to measure the potency of measles in trivalent measles, mumps and rubella (MMR) vaccines using quantitative real-time PCR (qPCR). Vero cells were infected with serial dilutions of a trivalent vaccine or a trivalent reference with known potency. Virus was allowed to replicate and subsequently replicated virus was quantitated by qPCR using the LightCycler technology. The virus titer in vaccine samples was estimated against reference preparations using parallel line analysis. In comparison to the plaque assay, the qPCR infectivity assay was faster and less laborious, while accuracy and intermediate precision were similar.

摘要

减毒活病毒疫苗的效力是通过对细胞培养物中的活病毒进行计数或滴定来确定的。这些传统的效力测试存在耗时费力且实验室间差异较大的缺点。在本研究中，我们描述了一种使用定量实时聚合酶链反应（qPCR）来测量三价麻疹、腮腺炎和风疹（MMR）疫苗中麻疹效力的快速方法的开发和验证。用已知效力的三价疫苗或三价参考品的系列稀释液感染非洲绿猴肾细胞（Vero细胞）。使病毒复制，随后使用罗氏LightCycler技术通过qPCR对复制的病毒进行定量。使用平行线分析，根据参考制剂估计疫苗样品中的病毒滴度。与空斑试验相比，qPCR感染性试验更快且更省力，而准确性和中间精密度相似。