聚合酶链反应(PCR)的热性能对乙型肝炎病毒检测的影响

IMPACT OF THERMAL OERFORMANCE OF PCR ON THE TESTING OF HBV-DNA

聚合酶链式反应(PCR)是由变性、退火及延伸三个步骤反复循环的DNA体外扩增技术。本文实验研究了PCR仪的三个阶段的热性能。结果显示,PCR实际温度与程序设置温度之间存在着较大的偏差;在变性阶段的温度差达3K以上;而且处于平台温度±1℃范围内的时间也比设置的要短得多。本文同时用3.3 kb(扩增区150 bp)的乙型肝炎病毒(HBV-DNA)为试验材料(模板),实验研究了变性温度及变性时间对PCR扩增结果的影响。用电泳法分离PCR产物结果表明:当变性温度低于91℃,变性温度高于95℃,或者变性时间超过30 s都会导致乙型肝炎病毒PCR检验结果的假阴性。

By PCR （the polymerase chain reaction） technique, DNA is amplified in vitro by a serious of polymerization cycles. A PCR cycle consists of three thermal steps： denaturation, annealing and extension. The experiment shows that there is quite large deviation between the program step and the actual step. The temperature deviation during the denaturation is larger than 3 K; the running time at the plateau at the temperature of 90℃±1℃ is quite short. At the same time, HBV-DNA is selected as a template, and the impact of the denaturation temperatures and the denaturation time of PCR procedure is studied. The products are tested by the electrophoresis. False negative may be produced if the denaturation temperature is lower than 91℃ or higher than 95℃, or the denaturration time is longer than 30 s.