聚合酶链式反应变性温度与产物假阴性的研究

Study of temperature and false negative products of polymerase chain reaction

背景:虽然聚合酶链反应方法具有特异性强、灵敏度高、操作简单、快速的特点,但同时也发现用聚合酶链反应技术进行生物化学实验和医学检验时,经常会出现假阴性的现象。目的:探讨聚合酶链反应变性温度与产物假阴性关系。设计、时间及地点:观察实验,于2008-03在吉林大学第二医院中心实验室完成。材料:重组质粒pUC-19(含有HBV完整基因组)和大肠杆菌JM109由吉林大学第二医院中心实验室提供。质粒DNA小量抽提试剂盒购自爱思进AXYGEN生物技术(杭州)公司。方法:以含有HBV完整基因组的重组质粒pUC-19为模板进行聚合酶链反应扩增,人为改变聚合酶链反应循环变性温度,观察其对聚合酶链反应扩增结果的影响。主要观察指标:以不加模板的反应体系为阴性对照,以加2倍于正常反应体系的模板反应体系为阳性对照,肉眼在紫外灯下观察不到产物判为阴性。结果:随着变性温度的降低,聚合酶链反应扩增效率降低,产物量明显减少。当变性温度低至85℃及85℃以下,琼脂糖凝胶电泳肉眼观察不到扩增产物。结论:较低的变性温度可导致扩增效率降低甚至假阴性的结果。

BACKGROUND： Although polymerase chain reaction （PCR） is characterized by strong specificity, high sensitivity, simple operation, and rapid processing, it is still limited by false negative during biochemical and medical examiantion. OBJECTIVE： To explore the relationship between temperature and false negative products of PCR. DESIGN, TIME AND SETTING： An observational study was performed at Central Laboratory of the Second Hospital of Jilin University in March 2008. MATERIALS： Recombinant plasmid pUC-19 （containing intact genome of HBV） and Escherichia coil （E. coli） JM109 were provided by Central Laboratory of the Second Hospital of Jilin University; small-dosage plasmid DNA extracting kit was provided by AXYGEN Biotechnology Company, Hangzhou. METHODS： PCR was amplified on recombinant plasmid pUC-19 （containing intact genome of HBV）, and temperature was artificially changed to detect the effect on PCR amplification. MAIN OUTCOME MEASURES： Reaction system without template was considered as the negative control group; reaction system with template as twice as normal reaction system was considered as the positive control group. PCR products which were not observed under ultraviolet lamp were determined as the negative. RESULTS： When the temperature was decreased, PCR amplification efficiency was decreased, and PCR products were less. When the temperature for denaturalization decreased to 85℃ or even below, no visual PCR products were observed on agarose gel electrophoretic profile. CONCLUSION： The lower denaturation temperature can decrease amplification efficiency or even cause the false-negative results of PCR.