How To Transition From Liquid to Lyophilized PCR Assays

Evaluation of a Transition From a Liquid to Lyophilized PCR Assay Introduction Lyophilized PCR assays, or assays that have been freeze-dried for preservation and long-term storage, offer several advantages over liquid PCR assays, primarily due to their enhanced stability and extended shelf life. This eliminates the need for cold chain storage and logistics and makes lyophilized PCR assays particularly valuable for point-of-care testing and distribution to resource-limited settings. The ready-to-use format of lyophilized assays simplifies laboratory workflows, reducing preparation time and minimizes the risk of human error. These benefits collectively contribute to more reliable and efficient PCR testing across various applications. Transitioning to a Lyophilized Assay A robust and reliable PCR assay is typically developed through the optimization of interdependent reaction factors such as: • Amplification buffer formulations • Enzyme concentrations • MgCl2 concentration • Primer/probe design and concentrations • Thermal cycling instrumentation and thermal cycling conditions • Amplification reaction vessel choices (such as tubes or plates and the potential impact of those plastics) Unfortunately, some common reaction components in liquid PCR assays, such as glycerol or some PCR buffer additives, are not compatible with lyophilization. Consequently, transitioning to a lyophilized PCR assay often requires the reoptimization of reaction conditions using lyophilization-compatible components. Therefore, when considering a transition from a liquid to lyophilized assay format, using a standardized performance evaluation framework can help compare the performance of two amplification assay formats. Comparison of DNA Amplification Between Liquid and Lyophilized PCR Assays We compared PCR assay performance of the liquid GoTaq® Probe qPCR Master Mix (Cat.# A6101) with a lyophilization-compatible version of the GoTaq® Probe qPCR Master Mix that was then lyophilized into bead format. We used a performance evaluation framework to compare the PCR performance for specificity, repeatability, linearity, sensitivity and efficiency (Table 1). Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 WHITE PAPER 1 Lyophilization: • Preserves Activity • Enhances Stability • Simplifies Storage & Shipment • Improves Handling 2 Evaluation of a Transition From a Liquid to Lyophilized PCR Assay WHITE PAPER Procedure: 1. Preparation of PCR amplification plates. a. We prepared a bulk reaction mix of GoTaq® Probe qPCR Master Mix by combining the liquid 2X GoTaq® Probe qPCR Master Mix with primers, probes, and nuclease-free water for enough reactions (N+1) to amplify the standard curve in replicates. We then aliquoted this bulk reaction mix into the wells of a PCR plate. b. We prepared the lyophilized GoTaq® Probe qPCR beads by dispensing the bead into the wells of a PCR plate and rehydrating with primers, probes and nuclease-free water. 2. Preparation of a standard curve and addition of samples to amplification plate. We prepared a standard curve by serially diluting a highpurity stock of DNA sample template. We then aliquoted the diluted samples into the wells of the amplification plate containing the PCR mixes. 3. Amplification and analysis. We amplified reactions using primers and a probe specific to human RNase P3 , maintaining the same primer/probe concentrations and amplification cycling conditions. We evaluated the performance of the amplifications according to the criteria described in Table 1. Table 1. Evaluation Criteria for Amplification Performance. Performance Attribute Evaluation Criteria Specificity No (or very late) amplification in No Template Control (NTC) reaction. Repeatability Standard deviation of the Cq values for replicate samples is ≤0.5. Linearity The R2 of a linear regression equation for the standard curve samples is ≥0.98. Sensitivity What is the lowest DNA concentrated included in the linear range that does not negatively impact the R2 and the standard deviation of the Cq values for replicate samples is ≤0.5? Efficiency The PCR efficiency is between 90–100%. Prepare Bulk Reaction Mixes: Add template DNA sample to each well Primers + Probes + Water 2X GoTaq® Probe (liquid) qPCR Master Mix + Primers + Probes + Water Aliquot prepared bulk reaction mix to wells of amplification plate: Rehydrate lyophilized bead with primer/probe/water mix Lyophilized GoTaq® Probe Master Mix Bead (1 reaction) 3 Evaluation of a Transition From a Liquid to Lyophilized PCR Assay WHITE PAPER Results: The lyophilized version of the GoTaq® Probe PCR Master Mix performed equivalently to the standard liquid format (Figure 1). Specificity, repeatability, linearity, sensitivity and efficiency of the lyophilized reaction were all within acceptable ranges. Performance Attribute Results GoTaq® Probe qPCR Master Mix Lyophilized Equivalent Specificity PASS No NTC amplification PASS No NTC amplification Repeatability PASS All standard deviations for 0.08–50ng/µl were ≤0.5. PASS All standard deviation for 0.016–50ng/µl were ≤0.5. Linearity PASS R2 = 0.997 PASS R2 = 0.996 Sensitivity 0.08ng/µl 0.016ng/µl Efficiency PASS 96.5% qPCR efficiency PASS 98.4% qPCR efficiency Figure 1. Comparison of the GoTaq® Probe qPCR Master Mix and a lyophilized equivalent master mix. Serial dilutions of human genomic DNA were amplified via qPCR with primers and probe specific to RNase P(3) using either the GoTaq® Probe qPCR Master Mix (Cat.# A6101) or a lyophilized equivalent of the GoTaq® Probe qPCR Master Mix. Amplification reactions were prepared according to the protocol described above. All samples were cycled on a QuantStudio™ 6 Pro Real-Time PCR Instrument using standard cycling conditions. Samples amplified with each master mix were analyzed using different thresholds; thus Cq values should not be compared between the two datasets. Panel A. Cq values for standard curve samples amplified with the GoTaq® Probe qPCR Master Mix (N=6) and Cq values for standard curve samples amplified with the lyophilized equivalent master mix (N=6). Panel B. Amplification performance of each master mix evaluated according to the criteria listed in the protocol above(1,2). 20 22 24 26 28 30 32 34 36 38 0.01 0.1 1 10 100 Cq DNA Concentration (ng/µl) GoTaq® Probe Lyophilized Equivalent B. A. 4 PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330 www.promega.com•©2024 PROMEGA CORPORATION • ALL RIGHTS RESERVED • PRICES AND SPECIFICATIONS SUBJECT TO CHANGE WITHOUT PRIOR NOTICE • PRINTED IN USA 8/24 • MSLG-1019 • PART #WP135 Evaluation of a Transition From a Liquid to Lyophilized PCR Assay WHITE PAPER Lyophilize Your Assay Our PCR Optimization Kit gives you a head start in creating a lyophilized assay. The kit contains pre-qualified, lyophilization-ready amplification components that are used to perform a short series of experiments to identify the optimal buffer, MgCl2 , and enzyme concentrations specific to your PCR application. Once you’ve identified your optimal lyophilization-compatible master mix, our customer service group will work with you to produce a pilot lot of lyophilized PCR beads to verify assay performance. Upon verification, we transition to final production of your lyophilized beads under our ISO-certified quality systems, ensuring that we deliver exactly what you need when you need it. Learn more about our fit-for-purpose lyophilized amplification assays or Let’s Talk Custom to get started today. References 1. Bustin, S.A. et. al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem. 55, 1–12. 2. Teter, S. and Steffen, L. (2017) Real-Time qPCR: Guidelines for a Comparison of Reagent Performance. Promega Application Notes #AN299 3. Centers for Disease Control and Prevention (CDC) (2022). Test Procedure: Monkeypox virus Generic Real-Time PCR Test, Rev. 01. Retrieved from: https://stacks.cdc.gov/view/cdc/119661. *