RNA SNP Detection Method With Improved Specificity Based on Dual-competitive-padlock-probe  

基于双竞争挂锁探针改进RNA SNP检测特异性的方法

作　　者：ZHANG Qin-Qin LI Jin-Ze ZHANG Wei LI Chuan-Yu ZHANG Zhi-Qi YAO Jia DU Hong ZHOU Lian-Qun GUO Zhen 张琴琴;李金泽;张威;李传宇;张芷齐;姚佳;杜鸿;周连群;郭振(中国科学技术大学生物医学工程学院(苏州),合肥230026;中国科学院苏州生物医学工程技术研究所,苏州215163;苏州大学第二附属医院临床检验科,苏州215004)

机构地区：[1]School of Biomedical Engineering(Suzhou),University of Science and Technology of China,Hefei 230026,China [2]Suzhou Institute of Biomedical Engineering and Technology,Chinese Academy of Sciences,Suzhou 215163,China [3]Department of Clinical Laboratory,The Second Affiliated Hospital of Soochow University,Suzhou 215004,China

出　　处：《生物化学与生物物理进展》2024年第11期3021-3033,共13页Progress In Biochemistry and Biophysics

基　　金：国家重点研发计划(2023YFC2413202);国家自然科学基金(82372142,52275581,82327802);江苏省重点研发项目(BE2022739);中国科学院青年创新促进会(Y2022088);中国科学院仪器开发项目(ZDKYYO20210004);苏州市科技发展项目(SJC2021019,SSD2023012,SSD2023017)资助。

摘　　要：Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.目的RNA的单核苷酸多态性(single nucleotide polymorphism,SNP)与多种疾病和药物反应相关的蛋白质表达有关,因此对其检测具有重要意义。目前,splintR连接酶辅助方法是RNA直接检测的重要方法,但当连接酶的保真度不理想时,该方法的特异性将受到限制。本研究的目的是创建一种提高splintR连接酶检测RNA特异性的方法。方法本研究提出了一种双竞争挂锁探针(dual-competitive-padlock-probe,DCPLP)检测方法,不需要额外的酶或反应,可提高splintR连接酶的特异性。为了验证该方法,采用双竞争挂锁探针介导的滚环扩增(rolling circle amplification,RCA)对CYP2C9基因进行RNA SNP基因分型。结果通过双挂锁探针的竞争和链替换,SNP检测的特异性得到了很好的提高,非特异性信号降低了83.26%。通过引入合成RNA样品,实现了10 pmol/L~1 nmol/L的动态检测范围。并将临床样本应用于该方法进行性能评价,结果与qPCR方法的基因分型结果一致。结论本研究成功建立了一种高特异性的RNA SNP直接检测方法,为准确鉴定各类RNA提供了新的途径。

关 键 词：RNA single nucleotide polymorphism GENOTYPING rolling circle amplification dual padlock probe 

分 类 号：Q522[生物学—生物化学] O65[理学—分析化学]