摘要
目的:建立和验证用于巨细胞病毒(CMV)启动子序列检测的复合实时定量 PCR 方法。方法以 CMV 启动子序列和小鼠管家基因β肌动蛋白基因的 cDNA 序列为模板分别设计探针和引物,用 SYBR GreenⅠ熔解曲线分析引物扩增的特异性。对反应体系进行系统优化,并验证方法的灵敏度、线性和重复性。结果针对 CMV 启动子序列的上游引物为5′AGACTTGGAAATCCCCGTGAGT3′,下游引物为5′CG-TATTAGTCATCGCTATTACCATGGT3′,探针为5′AACCGCTATCCACGCCCATTGATG3′。针对β肌动蛋白基因的上游引物为5′CCTGAGGCTCTTTTCCAGCC3′,下游引物为5′TAGAGGTCTTTACGGATGT-CAACGT3′,探针为5′TCCTTCTTGGGTATGGAATCCTGTGGC3′。用 CMV 启动子标准品制作的标准曲线,反应效率为100%,相关系数为0.9978,定量范围达到1.5×102~1.5×107拷贝,反应体系灵敏度为30拷贝。结论建立了检测 CMV 启动子序列的复合实时定量 PCR 方法。
OBJECTIVE To establish and validate a multiplex real time quantitative PCR method for cyto megalovirus(CMV)pro moter nucleic acid sequence detection.METHODS Probes and primers were designed according to CMV pro moter sequence and mouse β-actin house-keeping gene,the a mpli-fication specificity was analyzed using SYBR Green I dissociation curve.The reaction syste m was opti-mized,the sensitivity,linearity and reproducibility of the method were validated.RESULTS Forward primer sequence for CMV pro moter sequence were 5′AGACTTGGAAATCCCCGTGAGT3′;reverse prim-er sequence were 5′CGTATTAGTCATCGCTATTACCATGGT3′;probe sequence were 5′AACCGC-TATCCACGCCCATTGATG3′. Forward primer sequence for β-actin gene were 5′CCTGAG-GCTCTTTTCCAGCC3′; reverse primer sequence were 5′TAGAGGTCTTTACGGATGTCAACGT3′;probe sequences were 5′TCCTTCTTGGGTATGGAATCCTGTGGC3′.Reaction efficiency of the CMV standard curve reached 100%, correlation coefficient reached 0.9978, quantification margin was between 1 .5 ×102 and 1 .5 ×107 copies,and sensitivity of the reaction reached 30 copies.CONCLUSION The multiplex method that could absolutely quantify the copies of CMV pro moter sequence is established.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2014年第2期296-301,共6页
Chinese Journal of Pharmacology and Toxicology
基金
国家"重大新药创制"科技重大专项(2012ZX09302001)~~