中国药理学与毒理学杂志
中國藥理學與毒理學雜誌
중국약이학여독이학잡지
CHINESE JOURNAL OF PHARMACOLOGY AND TOXICOLOGY
2014年
2期
296-301
,共6页
苗玉发%王三龙%周晓冰%霍艳%耿兴超%吕建军%汪巨峰%李波
苗玉髮%王三龍%週曉冰%霍豔%耿興超%呂建軍%汪巨峰%李波
묘옥발%왕삼룡%주효빙%곽염%경흥초%려건군%왕거봉%리파
实时定量 PCR%CMV启动子%β肌动蛋白基因
實時定量 PCR%CMV啟動子%β肌動蛋白基因
실시정량 PCR%CMV계동자%β기동단백기인
real time quantitative PCR%CMV promoter%β-actin gene
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。
目的：建立和驗證用于巨細胞病毒（CMV）啟動子序列檢測的複閤實時定量 PCR 方法。方法以 CMV 啟動子序列和小鼠管傢基因β肌動蛋白基因的 cDNA 序列為模闆分彆設計探針和引物，用 SYBR GreenⅠ鎔解麯線分析引物擴增的特異性。對反應體繫進行繫統優化，併驗證方法的靈敏度、線性和重複性。結果針對 CMV 啟動子序列的上遊引物為5′AGACTTGGAAATCCCCGTGAGT3′，下遊引物為5′CG-TATTAGTCATCGCTATTACCATGGT3′，探針為5′AACCGCTATCCACGCCCATTGATG3′。針對β肌動蛋白基因的上遊引物為5′CCTGAGGCTCTTTTCCAGCC3′，下遊引物為5′TAGAGGTCTTTACGGATGT-CAACGT3′，探針為5′TCCTTCTTGGGTATGGAATCCTGTGGC3′。用 CMV 啟動子標準品製作的標準麯線，反應效率為100％，相關繫數為0．9978，定量範圍達到1．5×102～1．5×107拷貝，反應體繫靈敏度為30拷貝。結論建立瞭檢測 CMV 啟動子序列的複閤實時定量 PCR 方法。
목적：건립화험증용우거세포병독（CMV）계동자서렬검측적복합실시정량 PCR 방법。방법이 CMV 계동자서렬화소서관가기인β기동단백기인적 cDNA 서렬위모판분별설계탐침화인물，용 SYBR GreenⅠ용해곡선분석인물확증적특이성。대반응체계진행계통우화，병험증방법적령민도、선성화중복성。결과침대 CMV 계동자서렬적상유인물위5′AGACTTGGAAATCCCCGTGAGT3′，하유인물위5′CG-TATTAGTCATCGCTATTACCATGGT3′，탐침위5′AACCGCTATCCACGCCCATTGATG3′。침대β기동단백기인적상유인물위5′CCTGAGGCTCTTTTCCAGCC3′，하유인물위5′TAGAGGTCTTTACGGATGT-CAACGT3′，탐침위5′TCCTTCTTGGGTATGGAATCCTGTGGC3′。용 CMV 계동자표준품제작적표준곡선，반응효솔위100％，상관계수위0．9978，정량범위체도1．5×102～1．5×107고패，반응체계령민도위30고패。결론건립료검측 CMV 계동자서렬적복합실시정량 PCR 방법。
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.
