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目的研究和建立以细胞为基础的结合钠离子荧光探针检测雪卡毒素的方法,为快速、敏感检测雪卡毒素提供科学的实验依据。方法采用雪卡毒素标准品(P-CTX-1)结合乌苯苷和黎芦定处理小鼠脑神经瘤细胞(N-2A),用钠离子荧光探针(CoroNaTM Green)标记细胞,利用多功能酶标仪检测荧光强度,建立雪卡毒素浓度与荧光强度的标准曲线关系式;同时,采用CCK-8法和酶标仪检测毒素处理组细胞的存活率,建立细胞毒性实验的标准曲线关系式;分析比较两种方法的灵敏度。以两种方法分别对采集的33份深海珊瑚鱼样品进行毒素检测,对检测结果作分析比较。结果以细胞为基础的荧光检测法检测样品中毒素的检出限为10-13 g/ml,而细胞毒性试验检测法检测样品中毒素的检出限为10-12 g/ml,其结果具有一定的相关性,且荧光检测法灵敏度比细胞毒性试验高;同时钠离子荧光探针检测法的实验终点比CCK-8法缩短2～4 h。结论以细胞为基础的结合钠离子荧光探针检测法能快速、敏感地检测雪卡毒素,可作为雪卡毒素检测初筛方法推广应用。
OBJECTIVE To study and establish a cell-based method for detecting ciguatoxin by sodium ion fluorescence probe and provide a scientific experimental basis for rapid and sensitive detection of ciguatoxin. Methods The mouse neuroblastoma cells (N-2A) were treated with ciprofloxacin standard (P-CTX-1) and viprofloxacin and rivastigmine. The cells were labeled with sodium ion fluorescent probe (CoroNaTM Green) The relationship between the concentration of ciguatoxin and the standard curve of fluorescence intensity was established by the function of microplate reader. At the same time, CCK-8 method and microplate reader were used to test the survival rate of cells treated with toxin and the standard curve of cytotoxicity was established Type; analysis and comparison of the sensitivity of the two methods. Totally 33 samples of deep-sea coral reef fish samples were tested for their toxins by two methods. The test results were analyzed and compared. Results The detection limit of toxin was 10-13 g / ml in cell-based fluorescence detection and 10-12 g / ml in cytotoxicity assay. The result showed that The sensitivity of fluorescence detection method was higher than that of cytotoxicity test. At the same time, the end point of sodium ion fluorescence probe detection method was shorter than that of CCK-8 method by 2-4 hours. Conclusion Cell-based combined with sodium ion fluorescence probe detection method can rapidly and sensitively detect ciguatoxin and can be used as a screening method of ciguatoxin detection.