Bringing the heat: Thermostable analogs of Bst polymerase allow high-temperature LAMP.

Background: Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that gained prominence during the early months of the COVID-19 pandemic due to its simplicity, sensitivity and robustness. However, this technique is susceptible to non-specific amplifications, raising concerns about false-positive results and reduced diagnostic accuracy. A primary contributor to false-positive testing is primer dimerization, which can theoretically be mitigated by performing reactions at higher temperatures. Unfortunately, the strand-displacing DNA polymerases typically used in LAMP, such as Bst, exhibit reduced efficiency at elevated temperatures. To address this limitation, we hypothesised that naturally occurring thermophilic analogs of Bst may be capable of supporting LAMP at higher temperatures, thereby improving reaction specificity.

Methods: Bioinformatics and recombinant enzyme production allowed the identification and synthesis of several Bst analogs. These were tested in real-time LAMP assays to detect diverse targets, in a wide range of reaction temperatures (63°C-75°C) and in the presence of typical qPCR inhibitors.

Results: Three polymerases-Bst_7, Bst_8 and Bst_15-demonstrated exceptional activity and robust stability at higher temperature conditions (up to 72.5°C), while displaying considerable resistance to common qPCR inhibitors.

Conclusions: The identified thermophilic Bst analogs represent a potential solution for the mitigation of non-specific amplification in LAMP, further boosting the application of this technique in molecular diagnostic settings.