禾谷镰刀菌产毒分析及其毒素检测方法的研究
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摘要
小麦赤霉病是影响我国南方麦区、东北春麦区以及黄淮流域麦区的重要病害,可由多种镰刀菌引起,但主要致病种是禾谷镰刀菌Fusarium graminearum,通称为赤霉菌。赤霉菌引起小麦的穗腐,不仅使小麦籽粒产量降低、品质变劣,而且病麦粒中存留有病菌产生的真菌毒素,如脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)和玉米赤霉烯酮(Zearalenone,ZEN),食用污染毒素的病麦及其制成品后会引起人畜中毒,还有致癌、致畸和诱变的作用。但是我国相关的研究比较少,特别是检测水平偏低。因此,对我国禾谷镰刀菌产毒特点进行分析,并改进毒素检测方法,对我国麦制品中真菌毒素含量进行日常化的和标准化的检测,并发布相关毒素污染情况是非常必要的。
本研究从不同麦区分离并鉴定得到禾谷镰刀菌15个菌株,对其中6个产孢较好的菌株进行了产毒培养,并采用酶联免疫吸附法(ELISA)检测了这些菌株的DON和ZEN的产量。结果表明,不同菌株产DON的水平有明显差异,并得到了产DON较高的1个菌株,而各菌株产ZEN的水平都很低。
为建立基于单链抗体的毒素检测新方法,研究摸索了合成酶标抗原DON-HRP和ZEN-HRP的方法,得到了既保持毒素的抗原性又保持HRP活性的酶标交联物,可初步用于毒素的检测,但需进一步完善。
对从福建、江苏、四川、河南和黑龙江等麦区采集的55个小麦样品用ELISA法进行了DON和ZEN两种毒素含量的检测。结果有36个样品检测出DON,含量范围在0.1-5.2ppm,其中17个样品的DON含量超过国家限量标准1ppm;共有29个样品检测出ZEN,含量范围在0.3-228 ppb,其中3个样品的ZEN含量超过国家限量标准100ppb。病麦粒较多的小麦样品中毒素含量比较高,而无明显病症的小麦样品中毒素含量较低或检测不到毒素,说明病害程度与毒素含量之间存在一定的相关性;而且各样品中DON的含量均明显高于ZEN,说明禾谷镰刀菌以产DON为主。
本研究结果可以为进一步利用毒素筛选抗病突变体和抗性鉴定,以及为开发高效灵敏且成本低的检测试剂盒提供借鉴。
Fusarium head blight or wheat scab, caused by Fusarium graminearum (teleomorph Gibberella zeae (Schew) Petch), has been the most important disease of wheat, barley and corn in China. Head blight not only causes the severe grain yield loss, but also causes the harvested grain contaminated with several mycotoxins, such as deoxynivalenol (DON) and zearalenone (ZEN). The consumption of food product made from grains contaminated with these toxins is a potential problem for human and farm animals, which is known to cause carcinogenic, genotoxic acute or chronic diseases or to directly target the kidney, liver or immune system, and has caused great concern of food safety. However, the related studies on mycotoxins are fairly few, and the mycotoxin is not under regular detection in China. Therefore, it is necessary to study the characteristics of mycotoxin production, to improve the method of mycotoxin detection, furthermore to provide consumers with general information of mycotoxin contamination of agri-products and foods.Wheat grains were sampled from different wheat planting areas in China, including Fujian, Jiangsu, Sichuan, Henan and Heilongjiang provinces. Fifteen isolates of causal fungi from infected grains were purified and all were identified as F. graminearum with their morphological and molecular information, indicating that F. graminearum was the prevalent fungal species causing wheat scab in China. Among these isolates, toxin-production level was compared using enzyme-linked immunosorbent assay (ELISA) and it was revealed that there was obvious difference of toxin level within 6 isolates which produced conidia well, and 1 high toxin producing isolate was found.To explore the feasibility of mycotoxin detection by developing ELISA based on single chain variable fragment (scFv) of antibody, studies on conjugation of mycotoxin DON, ZEN with horseradish peroxidase (HRP) were conducted. Conjugates of both DON-HRP and ZEN-HRP kept the toxin antigenity and the HRP activity, suggesting they were feasible in application of ELISA kit development after more improvement.DON and ZEN of 55 wheat samples collected from different wheat planting areas in China were assayed with ELISA kit (Neogen Corp, USA). DON was detected ranging in 0.1-5.2ppm in 37 samples with 17 samples over 1ppm, the allowable limit of China National Food and Drug Standard. However, only 29 samples were contaminated with ZEN at fairly low amount of 0.3-228ppb, and there were 3 samples over 100ppb, the allowable limit of China National Food and Drug Standard. From the results, we also found that the samples which had high disease grain rate had high toxin contents; and vice versa, revealing that degree of disease and toxin contents were positively correlated. Moreover, DON contents in most wheat samples were much higher than ZEN contents, which showed that DON was the primary toxin produced by F. graminearum.In a summary, our study will facilitate screening scab-resistant wheat mutant and evaluating resistance of varieties with mycotoxin, and also make it possible to develop an easier ELISA kit based on scFv to detect mycotoxin at a lower cost.
本研究从不同麦区分离并鉴定得到禾谷镰刀菌15个菌株,对其中6个产孢较好的菌株进行了产毒培养,并采用酶联免疫吸附法(ELISA)检测了这些菌株的DON和ZEN的产量。结果表明,不同菌株产DON的水平有明显差异,并得到了产DON较高的1个菌株,而各菌株产ZEN的水平都很低。
为建立基于单链抗体的毒素检测新方法,研究摸索了合成酶标抗原DON-HRP和ZEN-HRP的方法,得到了既保持毒素的抗原性又保持HRP活性的酶标交联物,可初步用于毒素的检测,但需进一步完善。
对从福建、江苏、四川、河南和黑龙江等麦区采集的55个小麦样品用ELISA法进行了DON和ZEN两种毒素含量的检测。结果有36个样品检测出DON,含量范围在0.1-5.2ppm,其中17个样品的DON含量超过国家限量标准1ppm;共有29个样品检测出ZEN,含量范围在0.3-228 ppb,其中3个样品的ZEN含量超过国家限量标准100ppb。病麦粒较多的小麦样品中毒素含量比较高,而无明显病症的小麦样品中毒素含量较低或检测不到毒素,说明病害程度与毒素含量之间存在一定的相关性;而且各样品中DON的含量均明显高于ZEN,说明禾谷镰刀菌以产DON为主。
本研究结果可以为进一步利用毒素筛选抗病突变体和抗性鉴定,以及为开发高效灵敏且成本低的检测试剂盒提供借鉴。
Fusarium head blight or wheat scab, caused by Fusarium graminearum (teleomorph Gibberella zeae (Schew) Petch), has been the most important disease of wheat, barley and corn in China. Head blight not only causes the severe grain yield loss, but also causes the harvested grain contaminated with several mycotoxins, such as deoxynivalenol (DON) and zearalenone (ZEN). The consumption of food product made from grains contaminated with these toxins is a potential problem for human and farm animals, which is known to cause carcinogenic, genotoxic acute or chronic diseases or to directly target the kidney, liver or immune system, and has caused great concern of food safety. However, the related studies on mycotoxins are fairly few, and the mycotoxin is not under regular detection in China. Therefore, it is necessary to study the characteristics of mycotoxin production, to improve the method of mycotoxin detection, furthermore to provide consumers with general information of mycotoxin contamination of agri-products and foods.Wheat grains were sampled from different wheat planting areas in China, including Fujian, Jiangsu, Sichuan, Henan and Heilongjiang provinces. Fifteen isolates of causal fungi from infected grains were purified and all were identified as F. graminearum with their morphological and molecular information, indicating that F. graminearum was the prevalent fungal species causing wheat scab in China. Among these isolates, toxin-production level was compared using enzyme-linked immunosorbent assay (ELISA) and it was revealed that there was obvious difference of toxin level within 6 isolates which produced conidia well, and 1 high toxin producing isolate was found.To explore the feasibility of mycotoxin detection by developing ELISA based on single chain variable fragment (scFv) of antibody, studies on conjugation of mycotoxin DON, ZEN with horseradish peroxidase (HRP) were conducted. Conjugates of both DON-HRP and ZEN-HRP kept the toxin antigenity and the HRP activity, suggesting they were feasible in application of ELISA kit development after more improvement.DON and ZEN of 55 wheat samples collected from different wheat planting areas in China were assayed with ELISA kit (Neogen Corp, USA). DON was detected ranging in 0.1-5.2ppm in 37 samples with 17 samples over 1ppm, the allowable limit of China National Food and Drug Standard. However, only 29 samples were contaminated with ZEN at fairly low amount of 0.3-228ppb, and there were 3 samples over 100ppb, the allowable limit of China National Food and Drug Standard. From the results, we also found that the samples which had high disease grain rate had high toxin contents; and vice versa, revealing that degree of disease and toxin contents were positively correlated. Moreover, DON contents in most wheat samples were much higher than ZEN contents, which showed that DON was the primary toxin produced by F. graminearum.In a summary, our study will facilitate screening scab-resistant wheat mutant and evaluating resistance of varieties with mycotoxin, and also make it possible to develop an easier ELISA kit based on scFv to detect mycotoxin at a lower cost.
引文
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[5] Dixon D E, Ram B P, Hart L P, et al. Hybridoma cell line produces a specific monoclonal antibody to the mycotoxins Zearalenone and a-Zearalenol [J]. J. Agric. Food Chem., 1987, 35: 122—126.
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[9] 李群伟,王绍萍,鲍文生.真菌毒素与人类疾病的研究进展与展望[J].中国地方病防治杂志,2001,16(1):24—25.
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[12] Cromey M G, Shorter S C, Lauren D R, et al. Cultivar and crop management influences on Fusarium head blight and mycotoxins in spring wheat (Triticum aestivum) in New Zealand[J]. New Zealand Journal of Crop and Horticultural Science, 2002, 30: 235—247.
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