Ag^(+)/Cys介导的核酸探针级联荧光信号免疫检测法测定玉米和大米中黄曲霉毒素B_(1)

以葡萄糖氧化酶(GOx)催化Ag^(+)/Cys介导的富G-ssDNA/Tb^(3+)核酸探针体系引发荧光强度变化为方法学基础,开发了一种新型ELISA双重级联荧光信号放大系统,对葡萄糖氧化酶具有高的灵敏度响应(低至0.05μg/mLGOx)。将此体系与对GOx负载的纳米微球为信号探针的磁分离免疫体系相结合,建立了一种用于食品中AFB_(1)的高灵敏的双重级联信号放大的荧光免疫检测方法。该方法对AFB_(1)的检出限为0.05 ng/mL,在加标的玉米和大米样品中,添加回收实验结果表明,AFB_(1)的回收率在85.09%~105.2%之间,变异系数(CV)值为6.8%~15.7%。

Effects of Sterigmatocystin, Deoxynivalenol and Aflatoxin G_1 on Apoptosis of Human Peripheral Blood Lymphocytes in vitro

Objective To explore the effects of Sterigmatocystin (ST), Deoxynivalenol (DON) and Aflatoxin G1 (AFG1) on apoptosis of human peripheral blood lymphocytes (HPBLs) in vitro and thus to further elucidate the putative roles of these three mycotoxins on human immunosystem. Methods The effects of ST, DON and AFG1 on apoptosis of HPBLs were studied with cell culture, flow cytometric (FCM) DNA analysis and DNA agarose gel electrophoresis. Results DNA agarose gel electrophoresis results showed the characteristic 'ladder' pattern of apoptosis in HPBLs treated with ST, DON and AFG1. Flow cytometric DNA analysis revealed that typical subdiploid peaks of apoptosis in DNA histogram could be seen in all groups treated with the three mycotoxins. Significant time-effect and dose-effect relationships were found between the apoptosis rates and treatment time as well as concentrations of the three mycotoxins. Conclusion ST, DON and AFG1 can induce apoptosis of HPBLs in vitro and may have some negative effects on human immunosystem.

脱氧雪腐镰刀菌烯醇、黄曲霉毒素G_1对体外培养人外周血淋巴细胞凋亡影响的研究

以细胞培养、流式细胞仪ＤＮＡ含量分析及ＤＮＡ琼脂糖凝胶电泳方法研究了我国食管癌高发区磁县居民粮食中优势污染霉菌毒素脱氧雪腐镰刀菌烯醇（ＤＯＮ）和黄曲霉毒素Ｇ１（ＡＦＧ１）对人淋巴细胞凋亡的影响。流式细胞术（ＦＣＭ）检测结果显示，ＤＯＮ、ＡＦＧ１处理组淋巴细胞出现了典型的亚二倍体凋亡细胞峰，凋亡百分率与毒素作用时间（ＤＯＮ：２～７２小时；ＡＦＧ１：２～２４小时）及剂量（ＤＯＮ：５０～２０００μｇ／Ｌ；ＡＦＧ１：３．１２～２０００μｇ／Ｌ）呈正相关关系。ＤＮＡ琼脂糖凝胶电泳结果表明，ＤＯＮ（１０００μｇ／Ｌ）、ＡＦＧ１（１０００μｇ／Ｌ）处理２４小时淋巴细胞出现特征性的ＤＮＡ＂Ｌａｄｄｅｒ＂条带。因此表明，ＤＯＮ、ＡＦＧ１可诱导和促进体外培养的人外周血淋巴细胞发生凋亡。

黄曲霉毒素G_1与人血清白蛋白的结合机理及分子对接

在模拟人体血液p H 7.4的条件下,用分子对接及其荧光光谱、3D荧光、圆二色谱等方法研究黄曲霉毒素G1(aflatoxin G_1,AFG_1)与人血清白蛋白(human serum albumin,HSA)的相互作用。结果发现,根据双对数方程得出AFG1与HSA结合反应猝灭机制为静态猝灭,4个温度条件下结合常数数量级均为104,结合位点数近似为1。通过分子对接和热力学参数计算,AFG1结合在HSA的ⅠB疏水腔中,二者结合力主要为疏水作用和氢键。通过研究体内金属离子对AFG1-HSA反应的影响,Fe^(3+)、Mg^(^(2+))能增大AFG1对HSA的亲和力,而Zn^(2+)、Cu^(2+)、Mn^(2+)离子则能大大降低AFG1与HSA的亲和力。基于F?rster’s能量转移,二者反应距离为3.26 nm。3D荧光结果显示,二者的结合反应使HSA生色团氨基酸残基疏水性增加,二级结构发生改变;圆二色谱结果表明,加入AFG1使得HSA的α-螺旋含量增加。

Challenging the Expiry Dates of Two Mycotoxins Standards: Afla and Ochra A

Objectives: To challenge the expiry dates of low concentration high purity mycotoxins standards. Literature Review: Aflatoxins (AFs) and Ochratoxin A (OTA) are persistent mycotoxins with adverse effects on humans. Mycotoxins standards are purchased to determine mycotoxin concentrations in food and may be stocked in some laboratories beyond expiry dates causing laboratories financial losses. Methods: Certified mycotoxins standards were purchased over the years from the same supplier at times and at other times from two different suppliers for quality control purposes. For AFs, six chromatographic runs for each of the mycotoxins standards were done to compare the difference among these standards having the following expiry dates (2008, 2012, 2013 and 2018). AFs standards purchased/obtained from two different suppliers in 2016 and expiring in 2018 were also compared. For OTA, the difference of concentration obtained between two years (2010 and 2018) was tested. All samples were run on a HPLC equipped with a fluorescence detector. Linearity of calibration curves and the points of lowest detection were determined for AFs components and for OTA from the unexpired mycotoxins standards. Results: At a 0.05 significance level and using non parametric tests, the statistical test revealed a p of 0.166, 0.153, 0.358 and 0.03 for B1, G1, B2 and G2 respectively among years for standards from same supplier and 0.037, 0.109, 0.182 and 0.182 for B1, G1, B2 and G2 respectively for unexpired standards from two different suppliers. For OTA, a p of 0.109 was obtained for standards of different expiry dates purchased from different suppliers. Conclusion: High purity low concentration mycotoxin standards purchased a decade ago (i.e. expired) did not differ from those purchased this current year (still valid). Hence, the expiry date can be renewed reducing the laboratories expenses. Manufacturers are urged to reconsider the expiry dates.