基于气浴式核酸扩增的系统样本与空气温度延迟分析和优化

Analysis and Optimization of the Temperature Retard between Sample and Air Based on Nucleic Acid Amplification System Heated by Air

利用核酸扩增系统完成聚合酶链式反应(PCR)是特定基因片段进行扩增的主要途径。核酸扩增系统在升降温过程中反应样本与系统热源温度之间存在着较大延迟,这导致扩增过程中样本到达指定温度需要较长时间,降低了整体的反应速率;而且由于扩增过程易产生非特异性产物,样本温度长时间未到指定温度会增大非特异性产物生成率,降低反应的扩增效率。本文根据气浴式核酸扩增系统工作原理和Smart-cycler PCR仪特性设计了微型化气浴式核酸扩增系统,分析系统热量传递过程并计算样本空气热量传递时间,对实际扩增中样本与空气温度进行实时测量拟合温度曲线。依据测量和计算结果选择系统加热时间并通过改变空气温度记录不同温度下样本温度曲线,选择最优的样本温度曲线优化系统延迟。优化后扩增过程所需时间缩短为原来的50%,通过对单增李斯特菌进行扩增实验显示系统可在4min内完成3个扩增循环,扩增效果良好。优化样本与空气的温度延迟可以有效提高扩增速度和扩增效果。

It is the main method for amplifying the specific gene to use the nucleic acid amplification system to accomplish polymerase chain reaction （PCR）. The temperature retard between heat source and sample exists in the heating and cooling progresses of most nucleic acid amplification system. The retard would result in the problem that the sample would take a long time to reach the set temperature and the problem would reduce the speed of integrate reaction. Non-specific products would be created in the process of amplification when the sample cannot reach the set temperature within a certainly time and the amplified efficiency would be reduced. A miniaturization nucleic acid amplification system heated by air was designed in this study according to the principle of air-heated nucleic acid amplification system and the characteristics of the PCR instrument Smart-cycler. The heat transfer process was analyzed and the heat transfer time was calculated. The actual temperature was measured in real time, and the temperature curves were fitted. The heating time was chosen by analysis results and data fitting and the air temperature was changed, while the sample temperature was recorded. The retard between sample and air was optimized by choosing the best curve of sample temperature. The temperature retard between sample and air was reduced sharply and the required time of integrate progress is shortened to 50%. We confirmed from the amplification experiment of Listeria monocytogenes that the improved system could complete 3 cycles within 4 minutes, and the amplification effect was good. The amplification speed and effect could be improved effectively by optimizing the delay between sample and air.