Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Molecular Modification of Taq DNA Polymerase and Its Application in Probe-Based qPCR Direct Amplification System

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

As the key component of quantitative real-time polymerase chain reaction(qPCR)technology,Taq DNA polymerase’s performance directly affects the further development of qPCR technology.However,the wildtype Taq DNA polymerase has inadequate properties in inhibitor tolerance and elongation performance.To obtain Taq DNA polymerase with high performance,this study fused the double-stranded DNA-binding protein Sso7d or Sto7d to the N-terminal or C-terminal of wild-type Taq DNA polymerase by genetic engineering technology,which four soluble expression transformants were constructed,and then the better transformant was screened by tolerance test.The results show that the better transformant Taq-Sto has the highest tolerance,with no impact on its thermal stability,and the target can be successfully amplified by Taq-Sto under the extension condition of 1 s/kbp,indicating that Taq-Sto has enhanced extension performance.It also shows good tolerance to humic acid,tannic acid and whole blood in TaqMan qPCR system.EMSA experiment shows that the binding affinity of Taq-Sto to DNA tem⁃plate is improved,which is beneficial to enhancing the competitiveness of Taq-Sto to DNA template.Taq-Sto was applied to the TaqMan qPCR detection of African swine fever virus(ASFV).Compared with commercial reagents,Taq-Sto has lower detection limit of ASFV,and the detection sensitivity in 2%~6%(volume fraction)pig fecal samples or pork samples is 100.0%and 85.4%,respectively,indicating that Taq-Sto has more advantages in the field of direct qPCR detection.The results provide a reference for the development of DNA polymerase with better performance,which is conducive to further promoting the practical application of qPCR technology.