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

doi:10.12141/j.issn.1000-565X.230090

Abstract

Abstract:

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 wild-type 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 template 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.

Key words: Taq DNA polymerase, double-stranded DNA-binding protein, tolerance, polymerase chain reaction

CLC Number:

Q814

HU Songqing, YUAN Jiahui, LIU Guangyi, et al. Molecular Modification of Taq DNA Polymerase and Its Application in Probe-Based qPCR Direct Amplification System[J]. Journal of South China University of Technology(Natural Science Edition), 2024, 52(4): 8-16.

Figures/Tables 13

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模板质量浓度/（pg·μL^-1）	C_t
模板质量浓度/（pg·μL^-1）	Taq-Sto	商品试剂
112.00	29.04±0.06	28.83±0.13
28.00	31.45±0.12	31.40±0.15
7.00	33.52±0.20	34.50±0.01
1.80	35.86±0.90	36.53±0.69
0.44	36.41±0.03	39.14±0.32
0.11	38.71±0.52	40.28
阴性

猪粪样本添加量/%	C_t
猪粪样本添加量/%	Taq-Sto	商品试剂
0	28.89±0.01	28.24±0.04
2	30.36±0.10	28.48±0.02
4	29.86±0.22	28.91±0.14
6	29.93±0.22	31.14±0.24
8	29.69±0.18	38.40±0.88
10	30.62±0.32
12	30.94±0.45
14	33.62±0.37

猪肉样本添加量/%	C_t
猪肉样本添加量/%	Taq-Sto	商品试剂
0	28.36±0.03	28.49±0.10
5	28.77±0.05
10	29.23±0.10
15	30.25±0.14
20	31.29±0.15
25	32.90±0.26
30	36.51±0.37

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