RNA荧光定量标准品制备新方法的探讨  

Preparation of RNA Standards for Real-time Fluorescence Quantitative PCR

作　　者：黄竺筠[1] 张冬雷[2] 施建[2] 崔之础[2] 

机构地区：[1]南通大学附属医院心血管研究室,江苏南通226001 [2]南通大学附属医院酶学研究室,江苏南通226001

出　　处：《现代检验医学杂志》2005年第6期18-21,共4页Journal of Modern Laboratory Medicine

摘　　要：目的以丙型肝炎病毒(HCV)为例,介绍一种简便快速的RNA定量标准品的制备方法。方法RT-PCR扩增目的片段,产物以pGEM-T载体连接并转化感受态大肠杆菌DH 5α,经筛选和测序鉴定,阳性质粒体外转录合成cRNA,准确定量后作为标准品。结果RNA标准品具有较大的线性范围(109cop ies/m l^10 cop ies/m l),批内和批间重复性也较好,CV分别为1.68%~5.97%和6.40%~10.58%。结论此法制备的RNA标准品具有较好的检测灵敏度和特异性,适合临床推广应用。Objective To introduce a simple and quick method for preparation of RNA standard for real-time fluorescence quantitative PCR assay, HCV was used as an example. Methods Target gene was amplified by RT-PCR,ligated with pGEM-T vector,and transformed into competent E. Coli. DH5α. The plasmid identified by sequencing was transcribed in vitro,and complementary （cRNA） was quantified and used as standards. Results Standard curve created by RNA standards had wide detection range,and the coefficient of variation value for both intra-experimental and inter-experimental reproducibility ranged from 1.68% to 5.97%and 6.40% to 10.58% ,respectively. Conclusion It is useful of the RNA standards prepared by this method for clinical practice and spread.

关 键 词：RNA 标准品 定量 

分 类 号：Q503[生物学—生物化学]