端粒酶活性多重荧光定量PCR(Taqman探针)检测方法的建立及验证  

Development and verification of multiplex fluorescence quantitative PCR (Taqman probe) for detection of telomerase activity

作　　者：董莉 姜婷婷 周宇荀[1] 李凯[1] 肖君华[1] DONG Li;JIANG Tingting;ZHOU Yuxun;LI Kai;XIAO Junhua(School of Chemistry and Bioengineering,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学化学化工与生物工程学院,上海201620

出　　处：《中国生物制品学杂志》2023年第10期1218-1223,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31672550);国家重点研发计划(2018YFA081101)。

摘　　要：目的 建立端粒酶活性的多重荧光定量PCR(Taqman探针)检测方法,并进行验证。方法 针对端粒酶催化亚基(telomerase reverse transcriptase,TERT)的CDS序列设计特异性逆转录引物、2对定量引物及探针。优化反应体系中的逆转录引物(特异性逆转录引物和随机引物)及定量引物(2对定量引物探针单独及混合使用),与内参基因GAPDH引物和探针在单管中进行多重荧光定量PCR反应。用293T及MRC-5细胞分别制备端粒酶阳性标准品及阴性标准品,并验证方法 的稳定性及精密性。采用建立的方法 检测19份正常间充质细胞样本和32份乳腺癌细胞样本的端粒酶活性。结果 最佳反应体系为:以特异性逆转录引物合成的cDNA为模板,将TERT基因2对定量引物和探针混合后与内参基因GAPDH引物和探针在单管中进行多重荧光定量PCR反应。优化后大幅提高了体系的灵敏性,TERT荧光信号量(2Rn)增强了3倍。阳性标准品TERT基因扩增曲线正常,且与GAPDH基因之间的2Ct保持稳定;阴性标准品GAPDH基因扩增曲线正常,无TERT基因扩增曲线。反复冻融;3及5次阳性标准品的TERT与GAPDH基因之间的ΔCt与未冻融过的阳性标准品比较,差异较小;组内和组间精密性CV均<1%。19份正常间充质细胞样本端粒酶活性均为阴性,32份乳腺癌细胞样本端粒酶活性均为阳性,两者TERT基因的Ct值差异有统计学意义(t=4.236,P <0.001)。结论 建立的端粒酶活性多重荧光定量PCR(Taqman探针)检测方法 具有良好的稳定性及精密性,有望应用于肿瘤早期诊断和基因治疗。Objective To develop and verify a multiplex fluorescence quantitative PCR(Taqman probe) method for the detection of telomerase activity.Methods Specific reverse transcription primers,two pairs of quantitative primers and probes were designed for the CDS sequence of telomerase catalytic subunit telomerase reverse transcriptase(TERT).After optimization of the reverse transcription primers(specific reverse transcription primers and random primers) and quantitative primers(two pairs of quantitative primer probes used alone or in combination) in the reaction system,with the primer probe of internal reference gene GAPDH,multiplex fluorescence quantitative PCR was performed in a single tube.In addition,telomerase positive standard and negative standard were prepared with 293T and MRC-5 cells respectively,and the stability and precision of the method were verified.The telomerase activity in 19 normal mesenchymal cell samples and 32 breast cancer cell samples were detected by the developed method.Results The optimum reaction system was as follows:using cDNA synthesized with specific reverse transcription primers as the template,2 pairs of quantitative primer probes of TERT gene were mixed with internal reference gene GAPDH primer probes for multiplex fluorescence quantitative PCR reaction in a single tube.After optimization,the sensitivity and TERT fluorescence signal quantity of the system were greatly improved,and the ΔRn was enhanced by 3 times.The amplification curve of positive standard TERT gene was normal,and the ΔCt between TERT gene and GAPDH gene remained stable.The amplification curve of GAPDH gene in negative standard was normal,while there was no amplification curve of TERT gene.There was a little difference in ΔCt between TERT and GAPDH genes in the positive standard frozen and thawed for 3 and 5 times repeatedly and the positive standard without freezing and thawing,and the CVs of precision in intra-and inter-groups were all less than 1%.Telomerase activity was negative in 19 normal mesenchymal cell samp

关 键 词：端粒酶活性 多重荧光定量PCR法 端粒酶催化亚基 TAQMAN探针 

分 类 号：R34[医药卫生—基础医学]