B-241 Stabilization of Probe qPCR Mix: A Comparison of Capillary-Assisted Vitrification and Lyophilization

Abstract

Background qPCR master mixes are formulated for high specificity, sensitivity, and the reproducible diagnosis of diseases. However, traditional frozen master mixes have limitations, including strict transportation, storage, and usage conditions, which can impact their performance. Recent advancements in lyophilization (freeze-drying) have led to the creation of lyophilized bead or cake master mix products. Lyophilized qPCR master mixes offer benefits such as long shelf life and room-temperature storage, but they come with challenges. These include instability of certain components, the need for complex optimization, longer drying cycle, and increase costs. To tackle the challenges of lyophilization, we introduced capillary-assisted vitrification (CAV) processes, which can stabilize different biomolecules within an hour, with little to no optimization, and allow for storage at ambient temperature. In this comparison study, we evaluated the linearity, efficiency, analytical sensitivity, specificity, and stability of lyophilized and CAV-stabilized TaqMan probe qPCR mix. Methods The lyophilized PrimePath Probe qPCR mix was purchased from Takara Bio, stored at room temperature, and used as per the recommendation. For CAV sample preparation, 5X PrimePath Probe qPCR mix from Takara Bio was combined with Ambient BioFix buffer, applied to the Ambient BioFix scaffold, and desiccated for 30 minutes using the Ambient stabilizer. The stabilized mix was either used immediately or stored in Ambient storage bags. For stability testing, both lyophilized and CAV-stabilized samples were stored at 37°C for 2 months and 50°C for 1 month. On the testing day, lyophilized and CAV samples were rehydrated, and monkeypox virus (MPXV) primers, probes, and synthetic DNA were mixed and tested using the QuantStudio 5 Real-Time PCR System. Results To evaluate the performance of lyophilized and CAV-stabilized qPCR mixes, the linearity and efficiency of the stabilized reagents were tested using fivefold serial dilutions of MPXV DNA. Both reagents demonstrated similar coefficients of determination (R² = 0.99), with amplification efficiencies of 99% for the lyophilized sample and 105% for the CAV sample, both within the acceptable range for the assay. The analytical limit of detection (LOD) was 5 copies/reaction for the CAV sample and 10 copies/reaction for the lyophilized sample. As expected, both lyophilized and CAV-stabilized reagents showed undetermined cycle threshold (Ct) values when no template was used. Additionally, comparable amplification and Ct values (20 and 21) were observed for both lyophilized and CAV-stabilized samples when stored at 37°C for 2 months and 50°C for 1 month. Conclusion With little to no optimization and a one-hour cycle time, the qPCR mix was successfully stabilized using the CAV process without freezing. Its performance and stability were comparable to that of lyophilized mixes. The study recommends the CAV process as an alternative to complex lyophilization.