Preservation of primer and probes on “ready-to-use” 96-well microtiter plates: A step forward towards enhancing throughput and harmonization of real-time PCR applications in food and feed control

Three different agents, trehalose (TRH), gelatine (GEL) and polyethylene glycol (PEG) were investigated regarding their capability to preserve oligonucleotides, such as primer and probes or double-stranded plasmid DNA, pre-coated in 96-wells microtiter plates. The evaluation of the retained efficiency of selected multi-copy and two single-copy PCR (polymerase chain reaction) systems revealed best-suited final concentrations in the preservation mix of 1.5% (w/v) for PEG, 0.17% (w/v) for GEL and 0.07% (w/v) - 20/1 (TRH/nucleic acid w/w) - for TRH, respectively. Under these conditions PCR efficiencies did not deviate significantly from non-coated, freshly pipetted control reactions even after three months of storage at room temperature or under thermal stress at constantly 50 °C simulating an ageing effect. Among the three reagents TRH showed best performance since not only the most labile component - the fluorescent probe - is protected from degradation but also double-stranded plasmid DNA which can be used as reference material and in quantitative real-time PCR.

► A simple and efficient protocol for stabilization of DNA (primer, probes and plasmids) to be used in PCR was developed. ► Three different bio-protective agents (trehalose, gelatine and polyethylenglycol) were compared as stabilizing agents. ► A stabilizing effect was observed over a period of 3 month at 50 °C or at room temperature without loss in PCR performance. ► Trehalose in a concentration of 0.07% performed best.