Development of a duplex Proofman ladder-shape melting temperature isothermal amplification assay for simultaneous discrimination of Schisandra chinensis and Schisandra sphenanthera using species-specific ITS1 SNPs.

Traditional Chinese medicine (TCM) products frequently face adulteration risks, demanding rapid, field-deployable authentication methods. We developed a novel duplex Proofman ladder-shape melting temperature isothermal amplification (Proofman-duplex-LMTIA) assay that simultaneously discriminates Schisandra chinensis (authentic ingredient) from Schisandra sphenanthera (common adulterant) by targeting species-specific single nucleotide polymorphisms (SNPs) in the ITS1 region. Incorporating proofreading enzyme-mediated probe cleavage to enhance specificity, the assay uses dual fluorescence reporting (FAM/JOE) in a single closed-tube reaction at a constant temperature of 60 °C for 20 min. The duplex format achieved a detection limit of 10 fg/μL plasmid DNA for both targets, with no cross-reactivity against related plant species. It reliably detected adulteration levels as low as 0.1% (S. sphenanthera in S. chinensis) and 1% (vice versa) in binary mixtures. Validation using 13 commercial TCM products demonstrated 100% agreement with labeling. This isothermal method offers a practical, equipment-minimal molecular tool that complements chromatographic fingerprinting for on-site quality assurance in TCM supply chains.