Recombinase polymerase amplification-lateral flow (RPA-LF) assay combined with immunomagnetic separation for rapid visual detection of Vibrio parahaemolyticus in raw oysters

摘要

This study was the first attempt to optimize a recombinase polymerase amplification (RPA) and lateral flow (LF) assay combined with immunomagnetic separation (IMS) for the detection of Vibrio parahaemolyticus in raw oysters. The newly developed IMS-RPA-LF assay effectively combines sample preparation, amplification, and detection into a single platform. Under optimal conditions, the average capture efficiency (CE) for 104?colony forming units (CFU)/mL of four V. parahaemolyticus strains with 0.4?mg of immunomagnetic beads within 45?min was 80.3%. After optimization, the RPA-LF assay was able to detect V. parahaemolyticus within 15?min, comprising DNA amplification with RPA for 10?min at 37?°C and visualization of the amplicons through LF strips for 5?min. The RPA-LF assay exhibited good specificity by showing a test line for eight V. parahaemolyticus strains with different serotypes but no cross-reaction with 12 non-V. parahaemolyticus bacteria. RPA-LF assay was found to be sensitive and detected as low as 10?pg genomic DNA of V. parahaemolyticus. For spiked oyster samples, the detection sensitivity of V. parahaemolyticus was improved to 2?CFU/g by IMS-RPA-LF after enrichment for 4?h; in contrast, the IMS-PCR method required 8?h. Hence, even when V. parahaemolyticus was present in very low numbers in samples, the IMS-RPA-LF assay could be completed within half a workday. Because of the high sensitivity, specificity, and speed of the IMS-RPA-LF assay, this newly developed method opens a novel pathway for rapid diagnostic screening of V. parahaemolyticus in seafood, which is an increasingly important health issue worldwide.Graphical abstract