摘要
目的 建立一种实时、灵敏、特异的针对人／猴免疫缺陷病毒（SHIV）的RNA载量检测方法，通过与酶联免疫吸附试验（ELISA）进行比较，成为监测SHIV病毒体外复制过程的常用方法。方法 体外转录制备RNA标准品，利用TaqMan EZ逆转录聚合酶链反应（RT—PCR）试剂盒的反应体系和针对SHIVgag保守区91个碱基的Taq—Man探针与引物，建立一步法实时荧光定量RT—PCR。三种SHIV感染性分子克隆体外转染293T细胞，在不同的时间点采样，通过实时荧光定量RT—PCR和ELISA两种方法监测SHIV病毒复制过程。结果两种方法监测的病毒复制过程基本一致，转染后2～48小时病毒复制出现明显的对数生长期，之后进入平台期，p24浓度大约100pg／ml，而RNA载量大约在100拷贝／ml。两者具有很好的相关性（r^2=0．834；P〈0．001）。实时荧光定量RT＋PCR灵敏度更高，检测范围更广，费用更低。结论 所建立的一步法检测SHIV病毒载量的实时荧光定量RT—PCR方法，可以作为监测SHIV体外扩增的常用方法。
Objective To establish a real-time RT-PCR based plasma virus quantification method as a potential alternative tool to monitor SHIV replication in vitro by comparing with ELISA. Method Viral RNA standards were prepared by in vitro transcription and one-tube real-time RT-PCR was established and optimized using TaqMan EZ RT-PCR CORE REAGENTS and TaqMan probes,and primers directed to the 91 bases within the conserved gag region of SHIV. 293T cells were transfected with three kinds of SHIV infectious clones. Culture supernatants were sampled with two different kinds of style,and the virus concentration was tested by real-time RT-PCR and ELISA. Results There was no difference between the two methods in monitoring the course of virus replication, but real-time RT-PCR was more sensitive and cheaper,with wider range of detection.Conclusion The real-time RT-PCR could be used as an alternative method to monitor SHIV replication in vitro.
出处
《中国艾滋病性病》
CAS
2006年第4期300-303,310,共5页
Chinese Journal of Aids & STD
基金
CIPRA项目(U19AI051915)
973项目(2005CB522903)
