鼠艾滋病病毒荧光定量PCR标准品RNA的构建

Construction of Standard Friend Leukemia Virus RNA for Quantitative Fluorescent PCR

目的合成构建鼠艾滋病病毒(FLV)荧光定量检测标准品,明确核糖核酸(RNA)标准品体外合成的基本原则。方法通过序列对比,找到FLV最为保守的区段作为RNA标准品的合成目标,进行逆转录聚合酶链反应(RT-PCR)扩增,克隆于T载体,测序,设计带有T7启动子的引物,PCR扩增,将所得的PCR产物凝胶回收纯化,用T7RNA聚合酶进行体外合成RNA,最后用PCR和电泳检测验证。结果成功合成了FLV荧光定量PCR标准品。结论FLV荧光定量标准品的成功合成必须遵守以下4条基本原则:①选择保守序列;②设计引物时将潜在的终止密码或起始密码排除在PCR目标产物之外;③将启动子(如T7启动子)挂在适当的引物上,保证所合成的RNA模板与所要定量检测的RNA模板一致,而不是互补;④合成RNA后必须检测,以保证不含其他RNA或者未消化完的DNA。

Objective To synthesize standard friend leukemia virus （FLV） RNA for quantitative fluorescent PCR detection of FLV. Method The conserved FLV RNA region was amplified by reverse transcription polymerase chain reaction （RT-PCR）, then the PCR products were inserted into T-vector. Primers specific for T7 promoter were used for PCR amplification. Purified PCR products were then used the synthesis of standard FLV RNA. Result Standard FLV RNA was synthesized successfully. Conclusion Four basic principles were followed in this study： （1） selection of the conserved sequence; （2） both the start and stop codon should be excluded in the design of PCR primers for the target sequence ; （3） T7 promoter should be correctly added to the sense primer or anti-sense primer to ensure that the synthetic RNA is the same as the RNA for quantitative fluorescent PCR; （4） validation is required to ensure the synthetic standard RNA does not contain undigested DNA or other RNAs.