Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai, 200241, China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China.
Anal Bioanal Chem. 2020 May;412(12):2903-2914. doi: 10.1007/s00216-020-02532-9. Epub 2020 Mar 3.
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 10 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.
本研究首次尝试优化重组酶聚合酶扩增（RPA）和侧向流动（LF）检测与免疫磁珠分离（IMS）相结合，用于检测生牡蛎中的副溶血性弧菌。新开发的 IMS-RPA-LF 检测法有效地将样品制备、扩增和检测结合到一个单一的平台上。在最佳条件下，对于四种 V. parahaemolyticus 菌株，在 45 分钟内使用 0.4mg 免疫磁珠的平均捕获效率（CE）为 10 个菌落形成单位（CFU）/mL，达到 80.3%。经过优化，RPA-LF 检测法能够在 15 分钟内检测到 V. parahaemolyticus，包括在 37°C 下进行 10 分钟的 RPA 扩增和 5 分钟的 LF 条带可视化。该 RPA-LF 检测法具有良好的特异性，能够检测到 8 种不同血清型的副溶血性弧菌菌株，并且没有与 12 种非副溶血性弧菌细菌发生交叉反应。该 RPA-LF 检测法具有很高的灵敏度，最低可检测到 10pg 的副溶血性弧菌基因组 DNA。对于添加的牡蛎样品，通过 IMS-RPA-LF 富集 4 小时后，副溶血性弧菌的检测灵敏度提高到 2CFU/g；相比之下，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非常低，IMS-RPA-LF 检测法也可以在半个工作日内完成。由于 IMS-RPA-LF 检测法具有高灵敏度、特异性和速度，这种新开发的方法为海鲜中副溶血性弧菌的快速诊断筛查开辟了一条新途径，这是一个在全球范围内日益重要的健康问题。
