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.

中文翻译：

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重组酶聚合酶扩增-侧向流动（RPA-LF）分析与免疫磁分离相结合，可快速目测生牡蛎中的副溶血弧菌。

这项研究是优化重组酶聚合酶扩增（RPA）和侧向流动（LF）分析法与免疫磁分离法（IMS）结合以检测生牡蛎中副溶血弧菌的首次尝试。新开发的IMS-RPA-LF分析将样品制备，扩增和检测有效地结合到一个平台中。在最佳条件下，四种带有0.4 mg免疫磁珠的副溶血弧菌菌株在45分钟内对104个菌落形成单位（CFU）/ mL的平均捕获效率（CE）为80.3％。优化后，RPA-LF分析能够在15分钟内检测到副溶血性弧菌，包括在37°C下用RPA进行10分钟的DNA扩增，并通过LF试条对扩增子进行可视化5分钟。通过显示8 V的测试线，RPA-LF分析显示出良好的特异性。副溶血性菌株具有不同的血清型，但与12种非V型无交叉反应。副溶血性细菌。发现RPA-LF测定灵敏，检出的副溶血性弧菌低至10 pg基因组DNA。对于加标牡蛎样品，富集4 h后，IMS-RPA-LF将副溶血弧菌的检测灵敏度提高到2 CFU / g。相反，IMS-PCR方法需要8小时。因此，即使样品中副溶血性弧菌的数量很少，IMS-RPA-LF测定也可以在半个工作日内完成。由于IMS-RPA-LF测定的高灵敏度，特异性和速度，这种新开发的方法为快速诊断海鲜中的副溶血性弧菌开辟了一条新途径，这是世界范围内日益重要的健康问题。图形概要。