B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.