摘要
目的探讨脱氧尿嘧啶及尿嘧啶DNA糖基化酶抗PCR产物污染的能力。方法用Taq-Man荧光定量PCR检测方法进行定量检测。将一系列不同浓度的含dUTP的HBVvDNA PCR扩增产物作为污染源,分别加至含dUTP/UDC的PCR反应体系和普通荧光定量PCR反应体系中,在荧光定量PCR仪上进行定量检测,从而得出含dUTP/UDC的PCR反应体系的抗污染能力。结果最佳抗污染实验条件:UDG酶含量0.05 U,消化时间37℃5 min,灭活时间92℃1 min;dUTP100%代替dTTP。含0.05 U的UDG抗污染反应体系对每管浓度为103拷贝/ml的污染物可完全消化,并随UDC含量的增高及消化时间的延长,抗污染能力增强。结论dUTP/UDG反应体系的应用能有效防止PCR产物污染,但抗污染能力是有一定限度的,尚需加强实验室标准化管理,才能真正达到抗污染效果。
Objective To investigate the anti-contamination ability of the PCR reagent containing dUTP and UDG（dUTP/UDG system ）. Methods The different concentrations of the HBV-DNA PCR product containing 100% dUTP（ no dTTP） were added to the dUTP/UDG system. After digested by UDG for some time, then determined by the Lightcycler, and compared with the result of the primary, so as to get the anti-contamination efficiency of the dUTP/UDG system. Result The optimal experiment condition ： UDG 0. 05U, digestion time 37 ℃ 5min, inactive TM time 92 ℃ 1 min, and dTTP were totally replaced by dUTP. The dUTP/ UDG system could at least digest 103copies/ml PCR product of HBV-DNA. Conclusion The dUTP/UDG system can prevent the contamination of PCR product, but its efficiency of the anti-contamination is limited. The standardization of the lab is still very essential.
出处
《职业卫生与病伤》
2008年第2期67-70,共4页
Occupational Health and Damage
