应用单分子触发式级联指数恒温扩增技术检测长链非编码RNA方法的建立

Detection of long non-coding RNA by mono-molecule triggered cascade isothermal exponent amplification

目的建立一种利用核酸恒温指数扩增技术特异性检测长链RNA的高灵敏度检测方法。方法利用恒温指数扩增(EXPAR)反应对所建立的方法进行反应条件的优化,并对该方法的通用性、特异性和灵敏度进行了系统分析。结果单分子触发反应产物可以作为引物触发指数恒温扩增;柔性引物可以提高检测特异性,柔性臂长7nt;单分子触发式级联指数恒温扩增技术能够有效检测出待检样品浓度在10fmol/L^100pmol/L范围内的靶标RNA,线性响应良好(R 2=0.955)。结论单分子触发级联式恒温指数扩增技术可以快速、准确检测长链RNA。

Objective To establish a high sensitivity method for the specific detection of long-chain RNA by isothermal exponent amplification of nucleic acid. Methods In the study, the reaction conditions of the method were optimized by EXPAR reaction, and the universality, specificity and sensitivity of the method were analyzed systematically. Results The products of single molecule trigger reaction were successfully used as the primer to trigger the isothermal amplification. The primer with 7 nt spacer improved the specificity. The method of mono-molecular triggered cascade isothermal exponent amplification detected effectively the target RNA in the range of 10 fmol/L^100 pmol/L, and a good linear relationship was obtained(R2 =0.955). Conclusion The present method of mono-molecular triggered cascade isothermal exponent amplification would provide technology support for rapid detection of long-chain RNA.