Quantitative and rapid on-site identification of multi-species toxic mushrooms with a portable sample-to-answer platform

Mushroom poisoning presents significant food safety and public health risks. The rapid, accurate point-of-care testing is critical for detoxification and saving lives. However, traditional toxin-based assays require costly instrumentation and specialized operators, making them unsuitable for rapid on-site testing of poisonous mushrooms. Thus, we developed a portable multiplex qPCR platform featuring a novel nucleic acid release rubbing tube that enables integrated sample-to-answer molecular diagnosis. This mechanically assisted nucleic acid release concept eliminates the need for chemical lysis or centrifugation. The cost-effective platform detects as few as 0.5 copies µL -1 (830.5 zM or 1.54 ag/μL) and simultaneously tests 6 samples / 12 species in 38 minutes—nearly half the time of standard commercial mushroom qPCR assays. Assay specificity was validated using 8 primer/probe sets derived from database-scale internal transcribed spacer (ITS) regions design workflow. Moreover, we successfully tested 24 toxic mushroom samples under raw, cooked, and digested conditions, demonstrating robust adaptability across real-world matrices. Beyond mushroom toxins, this modular, field-deployable qPCR architecture provides a prospective framework for universal on-site pathogen detection, environmental surveillance, and resource-limited diagnostic applications. • Portable qPCR multiplex platform for on-site detection of toxic mushrooms. • Three-minute, one-step DNA release with rubbing tubes, no centrifugation required. • Database-scale eight species-specific primers design validated against 26 species. • Robust and high sensitivity/specificity in raw, cooked, and digested samples.