Exposure humidity and time control of lyophilization and enzyme engineering for stable isothermal nucleic acid detection reagents

Isothermal nucleic-acid testing is ideal for on-site diagnostics, but large-scale lyophilization of reagents is hindered by three bottlenecks: costly in-situ capping of vials or PCR tubes, activity loss during non-in-situ transfer/sealing caused by ambient humidity and exposure time, and wild-type T7 RNA polymerase inactivation due to lyophilization-induced structural changes. We overcame these obstacles by optimizing the freeze-drying protocol, screening for robust enzyme variants, and refining the protectant recipe. Sealing at 15% RH prior to stoppering proved optimal; a lyophilization-tolerant T7 RNA polymerase mutant termed V5 was isolated; and a blend of 3%(w/v) PEG8000 plus 0.5%(w/v) mannitol markedly enhanced stability. The resulting cake contains 3%(w/w) residual moisture, reconstitutes in <1 s, and retains full activity after 13 months at 4 °C and for approximately 2 months at 25 °C. These specifications enable short-term, cold-chain-independent field deployment of isothermal nucleic-acid detection reagents.