摘要
目的筛选合适的端粒和内参引物,对比实时荧光定量PCR(qPCR)和数字PCR(dPCR)检测端粒长度之间的差异。方法通过查阅文献选择6对引物并进行筛选,通过qPCR和dPCR方法检测0.625 ng/μl~20.0 ng/μl 6个不同浓度人类基因组DNA的端粒长度,对二者的线性拟合程度、相关性、重复性、检测成本、检测效率、用样量等因素进行对比。结果选择Tel1和Tel2作为端粒引物,RPLP0-F和RPLP0-R作为内参引物。qPCR端粒产物Ct值为11.68~16.21,内参产物Ct值为21.21~26.74,所得相对端粒长度值为739.29~1478.58,dPCR端粒拷贝数平均浓度为71.60 copies/μl~5235.67 copies/μl,内参拷贝数平均浓度为396.40 copies/μl~9499.67 copies/μl,相对端粒长度值为0.18~0.55。qPCR和dPCR均表现出良好的线性关系,前者端粒和内参回归系数为r^(2)值分别为0.9952、0.9931,后者r^(2)值分别为0.9752、0.9965,2种检测方法的结果成正相关(r^(2)=0.8760)。qPCR 3次重复测量的相对标准偏差(RSD)为0.07%~1.05%,dPCR为2.13%~11.12%,表明qPCR具有更好的稳定性。此外,qPCR检测成本低,单次检测量大,耗时较短,而dPCR用样量较少。结论检测数量较多样本的端粒长度时,qPCR比较合适,检测DNA含量较少样本的端粒长度时,可考虑使用dPCR。
Objective By screening suitable telomere and internal reference primers,this paper aims to compare the difference between quantitative real-time polymerase chain reaction(qPCR)and digital PCR(dPCR)in detecting telomere length.Methods Six pairs of primers were screened and selected through literature review.The telomere length of human genome DNA at six different concentrations of 0.625 ng/μl-20.0 ng/μl was detected by qPCR and dPCR,respectively.The factors including linear fitting degree,correlation,repeatability,detection cost,detection efficiency and sample size were compared.Results Tel1 and Tel2 were selected as telomere primers,RPLP0-F and RPLP0-R were selected as internal reference primers.The Ct values of telomere products measured by qPCR were within 11.68-16.21,the Ct values of internal reference products were within 21.21-26.74,and the relative telomere length values were within 739.29-1478.58.The average concentration of telomere copy numbers measured by dPCR was within 71.60 copies/μl-5235.67 copies/μl;the average concentration of internal reference copy numbers was within 396.40 copies/μl-9499.67 copies/μl,and the relative telomere length value was within 0.18-0.55.qPCR and dPCR showed a good linear relationship in detecting telomere length,the regression coefficient(r^(2))of telomere and internal reference for qPCR was respectively 0.9952 and 0.9931,dPCR was respectively 0.9752 and 0.9965,and the results of the two methods were positively correlated(r^(2)=0.8760).The relative standard deviations(RSDs)of three repeated measurements of qPCR were within 0.07%-1.05%,and dPCR were within 2.13%-11.12%,indicating that qPCR has better stability.In addition,qPCR has lower detection cost,larger single detection volume,and shorter time consumption,while dPCR uses fewer samples.Conclusion qPCR is more suitable for detecting the telomere length in a large number of samples,and dPCR can be considered for the samples with less DNA content.
作者
查玉娥
陈圆圆
朱牧
刘娟
石莹
魏岚
CHA Yu-e;CHEN Yuan-yuan;ZHU Mu;LIU Juan;SHI Ying;WEI Lan(China CDC Key Laboratory of Environment and Population Health,National Institute of Environmental Health,Chinese Center for Disease Control and Prevention,Beijing 100021,China)
出处
《中国卫生检验杂志》
CAS
2023年第19期2305-2311,2315,共8页
Chinese Journal of Health Laboratory Technology
基金
中国疾病预防控制中心环境与健康相关产品安全所青年科学基金(2020YSRF_01)。
