基于链替代扩增-纳米金比色直观检测单链核酸方法的研究

目的]将链替代扩增技术与核酸修饰纳米金积聚变色的光学特性相结合,设计了一种新型的直观检测3′端暴露单链核酸的方法,实现对单链核酸的高灵敏度检测。方法]设计一条含有硫代修饰酶切位点的单链核酸（ZDNA）,其酶切位点5′端是能将核酸修饰纳米金变色的Linker序列,3′端是能与Target单链核酸3′端完全互补的H序列。当无Target存在时,Linker会充分暴露,能使核酸修饰纳米金积聚呈现紫色;但是当Target存在时,会与H序列完全互补,作为ZDNA的引物进入链替代扩增循环,形成的新链不断与Linker序列互补,不能使核酸修饰纳米金积聚呈现红色,从而间接检测了Target单链核酸。结果]通过一系列试验确定检测体系,具体为：40μl修饰纳米金溶液（0.52 nmol/L）加入10μl酶循环体系（ZDNA20 nmol/L,33 mmol/L Tris-HCl（pH值7.9）;10 mmol/L MgCl2;66 mmol/LNaCl;0.5 mmol/LdNTP;0.1 mg/ml BSA;0.05 U/μl klenow;1 U/μl Hinc II）,直观或紫外检测并绘制Target浓度与纳米金积聚程度的标准曲线,表明Target浓度在1~200 pmol/L的范围内呈现良好的线性关系,R2=0.946,最低检测限是1 pmol/L。结论]链替代扩增-纳米金比色检测单链核酸方法简便、直观、成本低,与传统修饰纳米金比色检测DNA方法相比,灵敏度提高了104倍。

Colorimetric Detection Single-stranded Nucleic Acid Method Based on Gold Nanoparticles and Strand Displacement Amplification

Objective]A novel visual colorimetric 3 ′exposed single-stranded nucleic acid detection method in vitro was developed by combininng strand displacement amplification and the distance dependent optical properties of nucleic acid modified gold nanoparticles so as to improve the detection sensitivity.Method] Single-stranded(ZDNA) containd a phosphorothioate restricition site.5′end of the site was the Linker sequence which was able to change the color of the DNA-modified gold nanoparticles,3′ end was the H sequence which could be completely complementary with the 3′end of the target single-stranded nucleic acid.When no target existed,the linker would be fully exposed,and DNA modified gold nanoparticles would be accumulated and present purple.But when the target existed,3′end of it would be completely complementary with the H series,as the primer of the ZDNA,into strand displacement amplification cycle.The new generation single-stranded nucleic acid of the cycle could hybrid with the Linker sequence.And DNA modified gold nanoparticles wouldn't be accumulated and present red.So the Target single-stranded nucleic acids could be detected indirectly.Result] The specific detection system was that 40 μl modified gold nanoparticle solution(0.52 nmol/L) was added to 10 μl enzyme recycling system(ZDNA 20 nmol/L,33 mmol/L Tris-HCl(pH=7.9);10 mmol/L MgCl2;66 mmol/L NaCl;0.5 mmol/L dNTP;0.1 mg/ml BSA;0.05 U/μl klenow;1 U/μl HincII),and then detect the sample by UV and describe the standard curve of the relationship between the Target concentration and the degree of gold nanoparticles aggregation.The linear range was between 1-200 pmol/L;R2 was 0.946,the detection sensitivity of the Target was 1 pmol/L.Conclusion] The method was simple,intuitive,low cost,and compared with the traditional colorimetric DNA detection methods which nucleic acid modified gold nanoparticles were used,sensitivity was increased 104 fold.