腹泻性贝类毒素的细胞F-肌动蛋白荧光检测法的建立

目的通过检测大田软海绵酸(OA)对HL-7702肝细胞的F-肌动蛋白的解聚作用,建立腹泻性贝类毒素(DSP)的荧光检测法。方法使用鬼比环肽标记F-肌动蛋白,多功能酶标仪检测荧光强度,通过荧光强度的变化分析样品中毒素的含量。比较荧光检测法和ELISA法对贝类样品的检测结果,分析所建荧光检测法的可靠性。结果 OA能明显破坏细胞F-肌动蛋白的聚合。随着作用浓度的增加,破坏程度也随之增加。在2.5～40 nmol/L范围内呈现明显的线性关系(R2=0.9931),检测限值可达到2.01μg/100g贝肉;进行样本检测时,加标回收率为92.76%～96.49%,并与ELISA法检测具有较好的线性相关(R2=0.830)。结论与现有的检测法相比,F-肌动蛋白荧光检测法具有较好的重复性和较低的检出限,可用于有毒贝类的筛查与检测。

A fluorimetric microplate assay for detecting diarrheic shellfish poisoning toxins

Objective To establish a new assay for detecting the cytotoxicity of diarrheic shellfish poisoning(DSP) toxins.The assay is based on the depolymerization of F-actin induced by okadaic acid(OA),which is one of main components of DSP toxins.Methods HL-7702 Liver cells were stained with Oregon Green-514 phalloidin as a fluorescent marker for F-actin.The change of the fluorescence of F-actin treated with OA was measured with a fluorimetric microplate reader.The detection results of this assay and ELISA were compared to evaluate the reliability of the assay.Results OA caused an increase of F-actin depolymerization in a dose-dependent manner.There was a linear relationship between the concentration of OA and the depolymerization of F-actin in the range of 2.5-40 nmol/L of OA in 24 hours(R2=0.993).The detection limit of the F-actin fluorescence assay for OA was 2.01 μg/100g muscles in shellfish extracts,and the recoveries were 90%-100%.The results of the fluorescence assay were consistent with other methods mentioned above(R2=0.830).Conclusion F-actin fluorescence assay was a promising method for the detection of OA in shellfish for its convenience,shortcut and sensitivity.