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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 the FLV fluorescent quantitative detection standard and to define the basic principles of in vitro synthesis of RNA standard. Methods The most conserved region of FLV was found to be the synthetic target of RNA standard by sequence comparison, amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned into T vector, sequenced and the primer with T7 promoter was designed , PCR amplification, the resulting PCR product was gel purified, T7 RNA polymerase in vitro synthesis of RNA, and finally PCR and electrophoresis test validation. Results FLV fluorescence quantitative PCR standards were successfully synthesized. Conclusion The successful synthesis of FLV fluorescence quantitative standards must comply with the following four basic principles: ① Select conservative sequences; ② Design primers to exclude potential stop codons or start codons from PCR target products; ③ Put promoters (such as T7 Promoter) on the appropriate primer to ensure that the synthesized RNA template is consistent with the RNA template to be quantitated, rather than complementary; ④ After RNA synthesis, it must be tested to ensure that it does not contain other RNA or undigested DNA.