摘要
尿嘧啶-DNA糖基化酶(UDG)作为生物体内的DNA修复酶系,能有效水解单链或双链DNA上的尿嘧啶,可用于PCR过程中残留污染物的清除.本研究以PCR预混液试剂盒作为检测工具,以鲤浮肿病和罗非鱼湖病的阳性核酸片段检测为例,探讨了巢式和荧光定量PCR检测时,UDG的抗污染效果.研究结果表明:(1)向PCR反应体系中添加200μmol/L的dUTP,可保证扩增产物中有足够的尿嘧啶掺入,且对扩增效率影响较小.(2)使用0.2 U/10μL的UDG浓度,可清除常规PCR中99.9%以上(多数大于99.95%)的阳性污染物,或清除荧光定量PCR(探针法)中96.5%以上的阳性污染物.继续增加UDG用量,则污染物清除效率的提高有限.本研究为特定条件下UDG对污染性模板的清除效果做了定量统计,能为该技术的具体应用与进一步优化提供实验依据.
Uracil-DNA glycosylases(UDG)are evolutionary,well-preserved DNA-repair enzymes in most organisms,which initiate the base excision repair pathway and remove uracil from DNA.During PCR,UDG can be used to prevent carryover contamination.In this study,several PCR Master Mix Kits were served as the testing regent for positive DNA fragments of carp edema disease and tilapia lake disease,in order to evaluate the contamination clearance effect of UDG in both nested PCR and qPCR assays.The results obtained showed that:(1)Adding 200μmol/L dUTP to the PCR system will ensure enough uracil incorporation in amplicon and showed a lesser affection on the amplification efficiency.(2)UDG concentration of 0.2 U/10μL degrades more than 99.9%(above 99.95%in most cases)of positive contaminations in conventional PCR,or more than 96.5%positive contaminations in qPCR(TaqMan).Further increase the amount of UDG will not escalate its efficiency.During the research,we have made quantitative evaluation on the anti-contamination effect of UDG under specific conditions,which are practical references for the application and optimization of such contamination control protocol.
作者
陈凡
丁能水
李奕雅
汪小东
许弟群
CHEN Fan;DING Nengshui;LI Yiya;WANG Xiaodong;XU Diqun(School of Biological Science and Biotechnology,Minnan Normal University,Zhangzhou,Fujian 363000,China;Fujian Aonong Biological Science and Technology Group Co.Ltd,Zhangzhou,Fujian 363000,China;School of Physical Education,Minnan Normal University,Zhangzhou,Fujian 363000,China)
出处
《闽南师范大学学报（自然科学版）》
2022年第1期84-92,共9页
Journal of Minnan Normal University：Natural Science
基金
福建省自然科学基金项目(2021J01998,2018J01583)
福建省中青年教师教育科研项目(JAT200314)。
关键词
PCR
UDG
抗污染
水生动物病毒
核酸片段
检测
PCR
UDG
anti-contamination
aquatic animal viruses
nucleic acid fragment
test
