PCR仪性能对检测结果的影响

Effect of PCR instrument performance on assay results

中文关键词:  聚合酶链反应  温度  准确性  均一性  恒温时间

英文关键词:polymerase chain reaction  temperature  accuracy  homogeneity  hold-time

中文摘要:

      目的 评价仪器本身性能对PCR检测结果的影响。方法 使用不同厂家和不同型号的PCR仪, 选取特定基因片段进行扩增实验, 通过人为调整PCR仪的设定温度, 探究PCR仪温度准确性, 样品分别置于仪器上可能出现温度差异的位置进行扩增, 探究PCR仪温度均一性, 并探究恒温时间对扩增结果的影响。结果 当反应程序整体温度调高或降低1~2 ℃时, PCR仪检测弱阳性样本循环阈值(cycle threshold, Ct)值有极显著性差异(P<0.01)。实验过程中恒温时间达不到设定要求, 扩增结果会出现极显著性差异(P<0.01), 甚至造成扩增失败。结论 研究结果提示实验室应通过PCR校准和检测过程的质量控制, 确保检测结果的质量。

英文摘要:

      Objective To evaluate the effect of the instrument performance on PCR results. Methods The amplification experiments were carried out on different PCR instruments of different manufacturers by selecting specific gene fragments and artificially adjusting the setting temperature of the PCR instrument to investigate the effect of temperature accuracy on the temperature accuracy of PCR instrument, the samples were placed at locations where temperature differences might occur on the instrument for amplification to explore the temperature uniformity of PCR instrument; and the effect of constant temperature time (hold-time) on the amplification results was also investigated. Results When the overall temperature of the reaction program was adjusted up or down by 1?2 ℃, there was a significant difference in the Ct values of the weakly positive samples detected by the PCR instrument. When the temperature was not set at the required time, the amplification results were significantly different, and even caused amplification failure. Conclusion The result shows that laboratories should ensure the quality of the test results through PCR calibration and quality control of the testing process.