A real-time fluorogenic recombinase polymerase amplification microfluidic chip (on-chip RPA) for multiple detection of pathogenic microorganisms of penaeid shrimp

Pathogenic microorganisms pose a significant threat to global shrimp production, often resulting in widespread reductions in production across continents. In this study, we developed a centrifugal microfluidic chip integrating real-time fluorogenic recombinase polymerase amplification (on-chip RPA) for the parallel detection of five important pathogenic microorganisms: acute hepatopancreatic necrosis disease-causing Vibrio spp. (VAHPND), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), and Enterocytozoon hepatopenaei (EHP). This method enabled the parallel analysis of six genetic markers from a single sample through a single pipetting operation for sample injection. It also allowed for the highly automated detection of up to four samples simultaneously using a chip format. The on-chip RPA reaction was performed in a reduced system with a volume as low as 5 μL at a temperature of 39 °C for 20 min. This assay achieved a limit of detection (LOD) of 10 copies/μL of the recombinant plasmid of the target genes. The coefficient of variation (CV) for the time to positive (Tp) value was less than 0.10, even using concentrations at the LOD for multiple pathogens. This indicates a robust reproducibility among replicates. The clinical sensitivity and specificity for detecting field shrimp samples were 96.4% and 100%, respectively, compared with polymerase chain reaction (PCR) combined with DNA sequencing. On-chip RPA assays allow for low reagent consumption, ease of use, accelerated analysis, the ability to analyze multiple samples and genetic targets, and applicability for on-site detection and routine monitoring of multiple pathogens in shrimp aquaculture.