实时聚合酶链反应：一种用于评估减毒活骆驼痘疫苗和水牛痘疫苗效力的快速工具。

Real time PCR: a rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines.

作者信息

Prabhu M, Siva Sankar M S, Bhanuprakash V, Venkatesan G, Bora D P, Yogisharadhya R, Balamurugan V

机构信息

Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital (Distt.), Mukteswar 263 138, Uttarakhand, India.

出版信息

Biologicals. 2012 Jan;40(1):92-5. doi: 10.1016/j.biologicals.2011.09.012. Epub 2011 Oct 20.

DOI:10.1016/j.biologicals.2011.09.012

PMID:22019252

Abstract

In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (-3.324) and (-3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID(50) method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines.

摘要

在本研究中，分别采用基于骆驼痘病毒（CMLV）和水牛痘病毒（BPXV）的C18L基因（编码锚蛋白重复蛋白）的SYBR Green和TaqMan实时荧光定量PCR（rt-PCR）技术，对骆驼痘和水牛痘减毒活疫苗的效力进行评估。在关键时间点收获感染了相应疫苗病毒的细胞，并进行各自的PCR检测。CMLV和BPXV收获的关键时间点分别为感染后36小时和30小时，分别根据（-3.324）和（-3.321）标准曲线的最大斜率确定。对8批骆驼痘疫苗和7批水牛痘疫苗进行评估，结果表明，通过各自的rt-PCR测定的滴度与传统的半数组织培养感染剂量（TCID50）方法具有良好的可比性。发现rt-PCR检测比终点稀释法相对更灵敏、特异和快速。因此，它们可以作为评估骆驼痘和水牛痘疫苗中活CMLV和BPXV颗粒的额外工具。