文献类型： 外文期刊

作者： Zhang, Linzhong ¹ ; Dong, Rui ¹ ; Wei, Shu ¹ ; Zhou, Han-Chen ¹ ; Zhang, Meng-Xian ¹ ; Alagarsamy, Karthikeyan ¹ ;

作者机构： 1.Anhui Agr Univ, State Key Lab Tea Plant Biol & Utilizat, Hefei, Anhui, Peoples R China

2.Anhui Agr Univ, Sch Sci, Hefei, Anhui, Peoples R China

3.Anhui Acad Agr Sci, Tea Res Inst, Huangshan, Anhui, Peoples R China

期刊名称：PLOS ONE （ 影响因子：3.24； 五年影响因子：3.788 ）

ISSN： 1932-6203

年卷期： 2019 年 14 卷 6 期

页码：

收录情况： SCI

摘要： Quantitative real-time polymerase chain reaction (qPCR) is routinely conducted for DNA quantitative analysis using the cycle-threshold (Ct) method, which assumes uniform/optimum template amplification. In practice, amplification efficiencies vary from cycle to cycle in a PCR reaction, and often decline as the amplification proceeds, which results in substantial errors in measurement. This study reveals the cumulative error for quantification of initial template amounts, due to the difference between the assumed perfect amplification efficiency and actual one in each amplification cycle. The novel CyC* method involves determination of both the earliest amplification cycle detectable above background ("outlier" C*) and the amplification efficiency over the cycle range from C* to the next two amplification cycles; subsequent analysis allows the calculation of initial template amount with minimal cumulative error. Simulation tests indicated that the CyC* method resulted in significantly less variation in the predicted initial DNA level represented as fluorescence intensity F-0 when the outlier cycle C* was advanced to an earlier cycle. Performance comparison revealed that CyC* was better than the majority of 13 established qPCR data analysis methods in terms of bias, linearity, reproducibility, and resolution. Actual PCR test also suggested that relative expression levels of nine genes in tea leaves obtained using CyC* were much closer to the real value than those obtained with the conventional 2(Delta Delta Ct) method. Our data indicated that increasing the input of initial template was effective in advancing emergence of the earliest amplification cycle among the tested variants. A computer program (CyC* method) was compiled to perform the data processing. This novel method can minimize cumulative error over the amplification process, and thus, can improve qPCR analysis.