Effects of storage conditions on the stability of qPCR reagents: implications for environmental DNA detection

• Published in: BMC research notes 2024-07, Vol.17 (1), p.199-7, Article 199
• Main Authors: Lopez, Mark Louie D, Crichton, Ellika M, Allison, Michael J, Dema, Anna H, Bonderud, Matthew T, Helbing, Caren C
• Format: Article
• Language: English
• Subjects: Chemical tests and reagents; Comparative analysis; DNA; DNA Primers - genetics; DNA, Environmental - analysis; DNA, Environmental - genetics; eDNA; Environmental aspects; Environmental DNA; Experiments; Extrachromosomal DNA; Freeze–thaw cycles; Freezing; Genetic research; Identification and classification; Indicators and Reagents; Polymerase chain reaction; Primer and probe stability; qPCR master mix; Reagents; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Research Note; Shelf life; Specimen Handling - methods; Storage; Storage conditions; Surveys; Synthetic DNA; Thermal properties
• ISSN: 1756-0500; 1756-0500
• DOI: 10.1186/s13104-024-06850-4

Environmental DNA (eDNA) detection is a transformative tool for ecological surveys which in many cases offers greater accuracy and cost-effectiveness for tracking low-density, cryptic species compared to conventional methods. For the use of targeted quantitative PCR (qPCR)-based eDNA detection, protocols typically require freshly prepared reagents for each sample, necessitating systematic evaluation of reagent stability within the functional context of eDNA standard curve preparation and environmental sample evaluation. Herein, we assessed the effects of long-term storage and freeze-thaw cycles on qPCR reagents for eDNA analysis across six assays. Results demonstrate qPCR plates (containing pre-made PCR mix, primer-probe, and DNA template) remain stable at 4 °C for three days before thermocycling without fidelity loss irrespective of qPCR assay used. Primer-probe mixes remain stable for five months of - 20 °C storage with monthly freeze-thaw cycles also irrespective of qPCR assay used. Synthetic DNA stocks maintain consistency in standard curves and sensitivity for three months under the same conditions. These findings enhance our comprehension of qPCR reagent stability, facilitating streamlined eDNA workflows by minimizing repetitive reagent preparations.