Abstract

Global trade faces increasing biosecurity threats from pests like mealybugs and plant viruses, yet conventional detection methods are usually time-consuming and lab-dependent, creating an urgent need for an efficient portable platform. This study developed a microfluidic chip-based assay to simultaneously identify two mealybug species (Planococcus minor and Dysmicoccus neobrevipes) and Orthotospovirus tomatomaculae (tomato spot wilt virus, TSWV). Species-specific primers targeting conserved genetic regions (28S rRNA, internal transcribed spacer ITS, and nucleocapsid protein gene) were designed. The assay achieved a limit of detection (LOD) of 103 copies/µL (based on plasmids) and 2.30 × 10−3 ng/µL (based on real samples) for P. minor, 104 copies/µL and 1.16 × 10−2 ng/µL for D. neobrevipes, and 103 copies/µL and 2.07 × 10−2 ng/µL for TSWV, with no cross-reactivity against non-target species and good producibility both intrassay and interassay. Parallel detection was also successful with complex nucleic acid mixtures. The clinical specificity and sensitivity was 100% and 92.31%. Integrated with isothermal amplification and microfluidic pattern, the quadruple-sector chip enables on-site screening of a maximum of eight samples within 30–60 min. This rapid, sensitive, and portable tool overcomes limitations of conventional phytosanitary methods, supporting real-time customs monitoring and risk-based biosecurity measures.

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