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

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.