Stability and performance comparison of capillary-assisted vitrification and lyophilization for probe qPCR master mixes

Abstract Recent advancements in freeze-drying (lyophilization) have led to the creation of lyophilized PCR mixes. The lyophilized cake or bead offers benefits compared to liquid formulations including extended shelf lives and room-temperature storage, but they come with a number of challenges. Lyophilization is incompatible with certain chemical compounds and biological materials limiting the range of components that can be co-formulated. Formulation optimization and long drying cycles are required, resulting in increased costs. To address these challenges, we developed capillary-assisted vitrification (CAV) (formerly called capillary mediated vitrification (CMV)) and reported its capability to rapidly stabilize different biomolecules without freezing, in a short period (1–3 h cycle time) and with improved thermal stability. In this study, we sought to evaluate the efficiency of CAV to stabilize probe qPCR mixes and to compare the performance of CAV-stabilized PCR mixes with lyophilized mixes using Monkeypox (Mpox) virus DNA as a template. Both CAV-stabilized and lyophilized samples observed similar coefficients of determination (R² = 0.99), with amplification efficiencies of 99% for the lyophilized and 105% for the CAV samples. The analytical limit of detection was 5 copies/reaction for the CAV sample and 10 copies/reaction for the lyophilized sample. Both stabilization reagents showed acceptable variability, with coefficients of inter-assay precision under 2.5%. As expected, CAV-stabilized and lyophilized reagents showed undetermined cycle threshold (Ct) values when no template control was used. Additionally, comparable amplification and Ct values were observed for both CAV-stabilized and lyophilized PCR mix when stored at 37 °C for 26 weeks and 50 °C for 4 weeks. In conclusion, CAV- stabilized PCR mix without freezing, in a shorter cycle, and its performance and stability were comparable to that of lyophilized reagent. The study recommends the CAV process could be an alternative to complex lyophilization for stabilizing PCR reagents.