Rational Design of a Novel DNA Polymerase From <i>Clostridium thermocellum</i> to Improve LAMP Detection Performance

Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection. Through bioinformatics analysis, the enzyme and the DNA-binding domain (DBD) from Pyrococcus abyssi were mutated for enhanced interaction between proteins and DNA. A chimeric mutant DBD_E146K-S738R reaches the detection threshold 13 min earlier than wild-type Cth DNA polymerase in real-time LAMP detection with a template concentration of 1.58 × 10⁵ fg/µL. It also showed the highest enzymatic activity at pH 9.0 and 65°C. The chimeric enzyme DBD_E146K-S738R exhibits good thermal stability, capable of performing LAMP reactions after treatment at 73°C or 70°C for 8 h. Moreover, it maintains high activity even under the inhibitory conditions of 50 U/mL heparin, 1.6 mM EDTA, 200 mM NaCl, 10% ethanol, 1.2 M urea, or 0.8% phenol. Notably, it was able to detect 1.58 × 10² ag/µL of the genome and 1.03 CFU/mL of the colony in Salmonella typhimurium detection. The enzyme's performance is superior to commercial Bst 2.0 and comparable to commercial Bst 3.0. The results suggest that DBD_E146K-S738R in LAMP exhibits great potential for molecular biological studies and clinical diagnostic analysis.