有复制能力的逆转录病毒的检测

Detection of Replication-Competent Retrovirus

运用逆转录病毒作为外源基因导入的载体，在实际临床运用时需要检测包装细胞在表达目的基因的同时，是否会产生有复制能力的逆转录病毒．本实验运用了Marker Rescue Assay(补救分析)和RT／PCR(逆转录PCR)两种方法。补救分析可检测到6×10^7CFU／ml(Colony Forming Units／m1)的有复制能力的逆转录病毒．RT／PCR的灵敏度为1CFU／10^3ml．这两种检测方法的建立，为逆转录病毒载体用于临床基因治疗的安全性提供一定的保证。

Retroviral vectors are the most used gene delivery vehicle in human gene therapy.When the therapeutic gene is packagedfrom producer cell line, one has to determined whether thereplication-competent retrovirus(RCR) is being generated. In this study, two methods, namely the marker rescue assay and RT/PCR, were employed to detect RCR in the cells. While the marker rescue assay can detect RCR with a limit at 6×102CFU/ml, RT/PCR can be used to detect RCR at as low as lCFU/103ml. Combining the specificity of the marker rescue assay and the sensitivity of RT/PCR, both assays together should serve as an adequate test for detecting the generation of RCR in retroviral producer cell lines.