Background: As a heat-resistant polymerase, Thermus thermophilus (Tth) DNA polymerase can be widely used in Polymerase Chain Reaction (PCR). However, its non-specific amplification phenomenon is serious, which greatly limits development. Objective: In this study, we prepared Tth monoclonal antibodies against Tth DNA polymerase and researched their application in hot-start PCR. Methods: Tth was recombinantly expressed and purified, and used as an antigen to immunize BALB/c mice to obtain monoclonal antibodies. The qualified monoclonal antibody and Tth are incubated for a period of time at a certain temperature to obtain the hot-start Tth. We tested the polymerase activity and exonuclease activity blocking performance of hot-start Tth. Finally, the hot start Tth was applied to one-step RT-PCR. Results: Tth with a purity of >95% was obtained, and ten monoclonal antibodies were obtained by immunization. After incubation, there were three monoclonal antibodies identified that could inhibit the polymerase activity of Tth at low temperature. Furthermore, these three antibodies have successfully eliminated non-specific amplification in practical applications. Conclusion: Three monoclonal antibodies were successfully validated. Among them, monoclonal antibody 9 has the best overall effect. They have the function of inhibiting at low temperature and releasing at high temperature, which can be used as Tth polymerase inhibitors in the field of molecular diagnostics.
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