Gravity-driven millifluidic platform for magnetic solid-phase extraction of Enterocytozoon hepatopenaei DNA from complex shrimp hepatopancreas.
Effective detection of Enterocytozoon hepatopenaei (EHP) in aquaculture is currently hindered by the lack of field-deployable extraction methods capable of processing complex, inhibitor-rich hepatopancreatic tissue. This study presents a gravity-driven millifluidic platform for the rapid extraction of EHP genomic DNA using an optimized, surfactant-compatible magnetic solid-phase extraction (MSPE) chemistry. Utilizing 5% PEG 8000 and 2.0 M NaCl, the platform facilitates the selectively capture of DNA from inhibitor-rich crustacean lysates. The 3D-printed device employs a tilting rocking plate to generate passive, gravity-driven flow, maintaining homogeneous magnetic bead suspension and maximizing solid-phase capture efficiency without external pumps. The integrated platform achieved a DNA yield of 2804.33 ± 15.31 ng/μL, a 5.9-fold increase over manual magnetic bead extraction. TaqMan quantitative PCR (qPCR) validation targeting the EHP SSU rRNA gene was developed. Using a standard curve spanning 101 to 107 plasmid copies (Ct = -3.611 log10 [copy] + 42.309, R2 = 0.998, amplification efficiency 89.2%), the on-chip MSPE achieved a validated analytical limit of detection (LOD) of 1 spore per reaction (100% detection rate, n = 21), whereas a commercial CTAB-based DNA extraction kit failed to achieve a validated LOD even at 10 spores (85.7%, 18/21). Nested PCR targeting the SWP gene was employed for field evaluation. A pilot study across two cohorts (N = 40) demonstrated consistent detection of confirmed EPH-positive cases; however, the small sample size precludes definitive diagnostic accuracy claims. With a total processing time under 30 min, this platform provides a high-efficiency extraction module. Future work will couple the device with isothermal amplification (e.g., LAMP or RPA) to realize a sample-to-answer system for resource-limited aquaculture.