锁核酸和不对称扩增辅助的等位基因多重引物识别KRAS基因突变的研究  

Study on the Multi-primer allelic discrimination analysis aided by locked nucleic acid and asymmetric amplification for detection of KRAS mutations

作　　者：师声 张静[1] 胡岳 刘义庆 褚福禄 兰文军[1] SHI Sheng;ZHANG Jing;HU Yue;LIU Yi-qing;CHU Fu-lu;LAN Wen-jun(School of Bioengineering,Qilu University of Technology(Shandong Academy of Sciences),Jinan 250353,China;Department of Laboratory Medicine,Shandong Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,China)

机构地区：[1]齐鲁工业大学(山东省科学院)生物工程学院,山东济南250353 [2]山东第一医科大学附属省立医院临床医学检验部,山东济南250021

出　　处：《齐鲁工业大学学报》2022年第5期55-61,共7页Journal of Qilu University of Technology

基　　金：校国际合作重点项目(QLUTGJHZ2018008)。

摘　　要：RAS突变的检测为指导结直肠癌的预测和预后提供了有力的工具。探讨一种简洁、低成本的用于检测KRAS基因突变的多重引物等位基因识别方法(multiplex primer allelic discrimination,MP_(m) AD)。利用不对称扩增和含锁定核酸(locked nucleic acid,LNA)的等位基因特异性引物、以及共同的反向引物和探针,进行多重孔检测和参考孔检测,以多重检测和参考检测的扩增循环数值差(ΔC_(q))来判定结果。同时使用福尔马林固定石蜡包埋组织(Formalin Fixed and Paraffin Embedded Tissues,FFPET)样本或在野生型基因组DNA背景下混合质粒进行非临床性能评价,并采用Kappa检验比较MP_(m) AD与Sanger测序的结果一致性。该方法对KRAS热点突变检测灵敏度至少为3%;凝胶电泳也验证了方法的特异性;重现性的变异系数(coefficient of variation,CV)<15%;同源基因检测未观察到交叉反应。通过分析115例FFPET标本,MP_(m) AD法与Sanger测序结果之间无统计学差异(k>0.9;p<0.001)。MP_(m) AD方法可以简洁、低成本地检测KRAS热点突变。Detection of RAS mutant provides a potent tool to guide predictive and prognosis in colorectal cancer(CRC).This study extends a concise and cost-effecitve multiplex primer allelic discrimination(MP_(m) AD)assay for detection of KRAS hot point mutants.To discriminate KRAS mutations,allele specific(AS)forward primers were decorated with locked nucleic acid(LNA)in multiplex amplification assay aided by asymmetric amplification,and co-hydrolysis probe and reverse primer were utilized in multiplex amplification assay and reference amplification assay,respectively.ΔC_(q)(differences in threshold cycles between the multiplex assay and the reference assay)was calculated to determine outcome.Non-clinical performance evaluation was conducted by using DNA from Formalin Fixed and Paraffin Embedded Tissues(FFPET)or plasmid blended in the background of wild-type genomic DNA.Kappa statistics were used to compare the method with Sanger sequencing.Data demonstrated that analytical sensitivities of KRAS were at least 3%.And PCR gel results validated the specificity.Coefficient of variation(CV)in reproducibility assessment was lessthan 15%.No cross-reactivity was observed in homologous genes assay.By analyzing 115 FFPET specimens,no statistically significant difference was found between the MP_(m) AD assay and Sanger sequencing(k>0.9;p<0.001).KRAS hot point mutants can be concisely and cost-effectively detected by using MPmAD assay described in this study.

关 键 词：多重引物等位基因识别 结直肠癌 锁核酸 RAS 

分 类 号：Q754[生物学—分子生物学]