优化反应体系提升Phanta DNA聚合酶对非处理标本的检测性能  

Dramatically enhancing direct detection from crude samples by Phanta DNA polymerase using optimized conditions

作　　者：陈振华 蔡甜 熊晓辉[2] 许文玲 郭晓博 任顺利 韩锦雄 张海如 CHEN Zhenhua;CAI Tian;XIONG Xiaohui;XU Wenling;GUO Xiaobo;REN Shunli;HAN Jinxiong;ZHANG Hairu(The People..s Hospital of Nanhai District,Foshan 528000,China;College of Food Science and Light Industry,Nanjing Tech University,Nanjing 211800,China;Nanjing Vazyme Biotechnology Co.,Ltd.,Nanjing 210034,China;College of Biology and Biotechnology,Wuhan Institute of Bioengineering,Wuhan 430415,China)

机构地区：[1]佛山市南海区人民医院,广东佛山528000 [2]南京工业大学食品与轻工学院,江苏南京211800 [3]南京诺唯赞生物科技有限公司,江苏南京210034 [4]武汉生物工程学院生命科学与技术学院,湖北武汉430415

出　　处：《生物加工过程》2018年第2期102-108,共7页Chinese Journal of Bioprocess Engineering

基　　金：江苏省重点领域共性技术攻关项目(苏经信科技(2016)636号)

摘　　要：在食品检测、法医鉴定、分子诊断等领域经常需要对未提纯DNA的非规范处理标本进行聚合酶链式反应(PCR)扩增。然而,由于非处理标本中存在多种抑制成分,常见的DNA聚合酶并不能满足非处理标本的检测需求。本研究通过调节添加剂、Mg^(2+)浓度以及酶量,对Phanta DNA聚合酶进行反应条件优化,提升Phanta DNA聚合酶突变体的扩增性能,并考察该聚合酶在优化条件下对血液、植物和食品等非处理标本的检测性能。结果表明:在反应缓冲溶液SF Buffer中加入质量分数5%二甲基亚砜(DMSO)和3 mmol/L Mg^(2+),以及50μL反应体系中使用1U酶时,Phanta DNA聚合酶突变体具有最强的扩增性能,可对低至0.05 ng基因组、植物叶片粗提液、高达40%全血浓度及食品粗提液进行有效检测,性能优于普通Taq DNA聚合酶及未进行反应条件优化的Phanta DNA聚合酶。本研究在食品检测、法医鉴定、分子诊断等应用中非处理标本的检测具有极大应用价值。Polymerase chain reaction were applied in food detection,forensic identification,molecular diagnosis,and so on.In these applications,lots of samples were crude samples without DNA purification.These samples had many inhibitors which would inhibit PCR reaction.To enhance direct DNA amplification from crude samples,like blood,plant,and food,reaction condition of Phanta DNA polymerase was optimized by PCR additives,gradient concentration and polymerase content.DMSO and magnesium were used as PCR additives and the optimal concentration was 5%DMSO and 3 mmol/L magnesium ion.The optimized reaction condition,in combination with 1 U Phanta DNA polymerase in 50μL reaction volum,enabled efficient detection of PCR products from as low as 0??050 ng human genome DNA,40%blood,leaf,and food crude extracts.Moreover,compared with commercial DNA polymerases,the novel PCR additives coordinate with Phanta DNA polymerase had better performance.This study has important application value of crude sample detection in food detection,forensic identification and molecular diagnosis.

关 键 词：PhantaDNA聚合酶 PCR 添加剂 粗品 快速检测 

分 类 号：Q814.9[生物学—生物工程]