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<p>Polymerase chain reaction (PCR) is an essential tool in research and diagnostics but is limited by the number of resolvable targets, reliance on target-specific probes, and assay-specific data interpretation. To overcome these challenges, we introduce Universal Signal Encoding PCR (USE-PCR), a novel approach combining universal hydrolysis probes, amplitude modulation, multispectral encoding, and standardized analysis for robust, scalable target detection. Using 32 synthetic templates, USE-PCR demonstrates a mean target identification accuracy of 92.6% ± 10.7% at high template copy and 97.6% ± 4.4% at low template copy, with linear correlation coefficients of 0.99 across four dPCR platforms and a dynamic range spanning four orders of magnitude. Integrating USE-PCR with RNase H-based detection chemistry enables 32 single nucleotide variants to be called simultaneously with up to 86.5% accuracy in cancer cell lines. Together, these results position USE-PCR as a transformative platform for high throughput, multiplexed analyte detection with applications in research and clinical settings.</p>

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Multiplexed, universal probe-based rare variant detection with USE-PCR

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月4日

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  • John Alvarado,
  • Lucien Jacky,
  • Dominic Yurk,
  • Aaron Aguiar,
  • Paul Belitz,
  • Jerrod J. Schwartz

摘要

Polymerase chain reaction (PCR) is an essential tool in research and diagnostics but is limited by the number of resolvable targets, reliance on target-specific probes, and assay-specific data interpretation. To overcome these challenges, we introduce Universal Signal Encoding PCR (USE-PCR), a novel approach combining universal hydrolysis probes, amplitude modulation, multispectral encoding, and standardized analysis for robust, scalable target detection. Using 32 synthetic templates, USE-PCR demonstrates a mean target identification accuracy of 92.6% ± 10.7% at high template copy and 97.6% ± 4.4% at low template copy, with linear correlation coefficients of 0.99 across four dPCR platforms and a dynamic range spanning four orders of magnitude. Integrating USE-PCR with RNase H-based detection chemistry enables 32 single nucleotide variants to be called simultaneously with up to 86.5% accuracy in cancer cell lines. Together, these results position USE-PCR as a transformative platform for high throughput, multiplexed analyte detection with applications in research and clinical settings.