A 3D-printed handheld device for quick citrus tissue lysis and nucleic acid extraction

Abstract

AbstractsA 3D-printed handheld device has been developed for rapid and efficient sample preparation from citrus leaves, aimed at streamlining protocols traditionally reliant on mortar and pestle. With its high-speed motor, knurled lysis chamber for rapid sample lysis, and quick nucleic acid extraction using paper disks, this device can yield ready-to-use extracts in just 12 minutes, significantly reducing the time required for sample preparation. The device was optimized for maximum sample lysis by evaluating operation voltages and chamber features. The results showed that the lysis chamber with internal knurling and the motor operated at 7.5 V was sufficient for effective sample lysis in 1 minute, achieving total RNA concentrations up to 87.6% of those obtained with a mortar and pestle. Furthermore, concerns regarding heat generation and resin release during the lysis process were found to not impact sample quality. To further facilitate in-field diagnosis, the capability of in-device sample preparation was verified with citrus sources infected with citrus tristeza virus and Spiroplasma citri in qPCR-based assays, where low assay variations were demonstrated (< 3.8%). Overall, the in-device sample preparation integrated with the paper disks showed good reliability and compatibility across different pathogens for downstream analysis. An eco-friendly sterilization protocol using household bleach and vitamin C solution was also developed to safely reuse the device for in-field deployment.