Vibrio parahaemolyticus isrecognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla_CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla_CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and other seven non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/L Mg²⁺, 0.96 mmol/L dNTPs, 4.8 U Bst DNA polymerase, 8:1 ratio of inner primer to outer primer, at 63 °C for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1×10^-4 ng/μL, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 cfu/mL, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples. It suggested that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

中文翻译：

---

使用基于 blaCARB-17 基因的环介导等温扩增和横向流动试纸 (LAMP-LFD)，快速和直观地检测水产食品中的副溶血性弧菌。

副溶血性弧菌被认为是导致人类肠胃炎的最重要的食源性病原体之一。bla _CARB-17基因是一种固有的β-内酰胺酶基因，是副溶血性弧菌的一种新的物种特异性遗传标记。在这项研究中，针对这种bla _CARB-17基因开发了一种环介导等温扩增 (LAMP) 测定法结合横向流动试纸 (LFD) 。LAMP-LFD 的特异性通过检测副溶血性弧菌ATCC 17802 和其他七种非副溶血性弧菌菌株来确定。最后，通过检测副溶血性弧菌污染，证实了 LAMP-LFD 的实用性样品和天然食品样品。结果表明，优化的LAMP反应参数为：2.4 mmol/L Mg ²⁺，0.96 mmol/L dNTPs，4.8 U Bst DNA聚合酶，内引物与外引物比例8:1，63 ℃ 40 分钟 LFD 检测的优化反应时间为 60 分钟。与七种非副溶血性弧菌菌株的交叉反应性分析表明，LAMP-LFD 对副溶血性弧菌具有特异性。LAMP-LFD对副溶血性弧菌基因组DNA的检测限为2.1×10 ^-4ng/μL，相当于 630 fg/反应，灵敏度比传统 PCR 高 100 倍。加标研究中的 LAMP-LFD 显示检测限约为 6 cfu/mL，这与传统 PCR 相似。开发的 LAMP-LFD 专门从 30 个海鲜样品中鉴定出 10 株副溶血性弧菌。这表明该 LAMP-LFD 可能是一种合适的诊断方法，用于检测水产食品中的副溶血性弧菌。