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基于腹泻性贝毒素(Diarrhetic shellfish poisoning,DSP)的致腹泻性组分大田软海绵酸(okadaic acid,OA)及其衍生物鳍藻毒素(dinophysistoxins,DTXs)能抑制蛋白磷酸酶活性的特点,建立了快速测定贝类中DSP的磷酸酶抑制比色分析方法。采用对硝基苯磷酸二钠(p-nitrophenyl phosphate disodium,p-NPP)为底物,其与蛋白磷酸酶2A(protein phosphatase 2A,PP2A)反应生成的黄色水解产物在碱性条件下于405 nm波长处有强烈的吸收峰,根据吸光度值计算抑制剂的浓度。酶抑制法继承了小鼠生物法能建立剂量-效应关系的优势,直接反映毒素的相对毒性大小,测定的是DSP毒素致腹泻性成分的总量,以OA浓度计。本研究优化了样品前处理方法并考察了基质浓度的影响。方法的筛选检出限为80μg/kg。采用该方法进行加标回收实验,回收率在90.43%~118.52%范围内,相对标准偏差(RSD)为6.85%~13.93%。该方法操作简便、快捷,回收率高,重现性好,可作为快速筛查工具用于贝毒的日常监控。
Based on diarrhea component diarrhetic shellfish poisoning (DSP), okadaic acid (OA) and its derivatives dinophysistoxins (DTXs) can inhibit the activity of protein phosphatase Phosphatase Assay Colorimetric Assay for Rapid Determination of DSP in Shellfish. Using p-nitrophenyl phosphate disodium (p-NPP) as a substrate, the yellow hydrolyzate reacted with protein phosphatase 2A (PP2A) under alkaline conditions at 405 nm Wavelength of a strong absorption peak, based on the absorbance value calculated inhibitor concentration. Enzyme inhibition method inherits the advantages of mouse biological method to establish the dose-response relationship, which directly reflects the relative toxicity of toxins, and determines the total amount of diarrhea-causing components of DSP toxin, measured by OA concentration. This study optimized the sample preparation method and investigated the effect of matrix concentration. The detection limit of the method was 80μg / kg. The method of spiked recovery experiments showed that the recoveries ranged from 90.43% to 118.52%, and the relative standard deviations (RSDs) ranged from 6.85% to 13.93%. The method is simple, rapid, high recovery rate, reproducible, and can be used as a rapid screening tool for the daily monitoring of shellfish poisoning.