关键词: 绝对定量qPCR, 质粒克隆, 标准曲线, 扩增效率, 拟合优度

Abstract: Real-time quantitative PCR （qPCR） is increasingly employed for absolute quantification of microorganisms. Appropriate standards and optimal protocols are the key to success. We constructed a plasmid which contained the complete 16S rRNA gene of a soil bacterium and could be amplified by four bacterial universal primer pairs targeting different 16S rRNA gene regions. Two primer pairs were used for absolute quantitative qPCR experiments. All melting curves were unimodal. The amplification efficiencies of the standard curve were 87.4% and 71.9%, and the fitting coefficients were all greater than 0.99. Three qPCR experiments were conducted within one day using the same gradient-diluted standards. The amplification efficiency and fitting coefficient were more stable with TE buffer or TE0.1 buffer for dilution, or at 4 ℃ storage. After 30 days of storage at －20 ℃, the amplification efficiency and fitting coefficient of the standard curve did not obviously decrease, but fluctuated at different times. Fluctuations could be reduced by using TE buffer or TE0.1 buffer for dilution. Stable storage and use of standards would enhance the accuracy and reproducibility of absolute quantitative qPCR experiments across different environments and times.

Key words: absolute quantitative qPCR, plasmid cloning, standard curve, amplification efficiency, goodness of fit