L-DNA calibrators for PCR amplicon characterization

While real-time polymerase chain reaction (PCR) is powerful on its own, its utility is markedly improved by post-PCR techniques like melt analysis and target quantification. We have previously utilized L-DNA stereoisomers matching the sequences of the reaction primers and target amplicon to increase PCR sensitivity and specificity in the presence of interferents. In this report we describe using L-DNA sequences as internal calibrators to confirm amplicon specificity and estimate initial target concentration. An S. mitis reaction with high analytical sensitivity was used to collect real-time PCR and melt data. In addition, melt data for end-labeled L-DNA and amplicon intercalator signal were acquired continuously during the PCR reaction and reinterpreted in post-PCR analysis. For melt analysis, melt curves were analyzed to produce a unitless melt signature via comparison to the L-DNA calibrator strands. These melt signatures were then converted to temperature using the different but known melt temperatures of the L-DNA calibrators. The S. mitis initial target concentration was estimated in three steps. First, the amplitudes of the melt peaks corresponding to the amplicon and the L-DNA high temperature calibrator were extracted from the melt data in each cycle. Second, the L-DNA melt derivative peak height was set as a known-concentration standard to convert intercalator fluorescence from the amplicon intercalator melt peak into amplicon concentration. Third, a sigmoidal model of efficiency decay estimated reaction efficiencies and initial S. mitis target concentrations from the amplicon melt derivative peak heights at later cycles. The S. mitis amplicon melt signature was found to be a consistent 0.8143 ± 0.0058 (mean ± std, n = 16) with a coefficient of variation of 0.7%. After translation to temperature, these values were equivalent to 84.96 ± 0.10 °C. Initial target concentration estimates were accurate above 10² copies/µL, with a coefficient of variation of 120% for 10² and a relatively constant 22% for the other concentrations. These proof-of-concept results suggest that the addition of L-DNA strands can serve as internal calibrators for single-sample characterization of a PCR amplicon.

The online version contains supplementary material available at 10.1186/s44331-026-00022-w.