稻谷中霉菌分析及aflR基因的SPR快速检测技术建立

发布时间：2018-01-13 21:37

本文关键词：稻谷中霉菌分析及aflR基因的SPR快速检测技术建立　出处：《湖南农业大学》2012年硕士论文　论文类型：学位论文

【摘要】：粮食安全是人们维持基本生活的重要保障,稻谷作为人们生活重要的粮食作物,其安全贮藏是重中之重。研究分析仓储稻谷中霉菌污染的状况,便于采取有效的措施来防止或降低污染；研究污染稻谷中的黄曲霉毒素生物合成的关键调控基因aflR与黄曲霉毒素之间的关系并建立SPR快速检测aflR基因的方法,有望在微生物未合成黄曲霉毒素前,检测到合成黄曲霉毒素的前体物质(aflR基因),起到预警的作用,杜绝黄曲霉毒素通过稻谷对人们身体健康的构成威胁及减少经济损失。本文以仓储稻谷为试验材料,采用经典微生物学分离纯化方法,分析稻谷中主要污染霉菌类型,通过ELISA与PCR相结合的方法筛选并鉴定了一株产黄曲霉毒素的菌株；利用RT-PCR方法克隆aflR基因,以及采用SPR生物传感器直接检测核苷酸技术,建立了SPR快速检测aflR基因的方法,得到以下结论： 1、以来自岳阳、坪塘地区仓储稻谷的13个样本为试验材料,分析稻谷中霉菌的数量均在105-106数量级之间,已经属于霉菌污染的较高水平；并且从形态学分析,污染的霉菌主要为曲霉属和青霉属。通过ELISA方法对污染频率较高的菌株进行黄曲霉毒素分析,发现2株产黄曲霉毒素的菌株。以产毒较强的菌株作为本试验的试验菌株,利用形态学、ITS rRNA序列分析相结合的方法对其进行了鉴定,确认该菌株为黄曲霉菌(Aspergillus flavus),命名为D1。 2、以A.flavus Dl为试验菌株,通过RT-PCR方法,克隆了与黄曲霉毒素产生相关的调控基因aflR基因。 3、利用DNA与DNA杂交的直接法,建立了SPR快速检测aflR基因的方法,检测标准探针的最低浓度为7.5nmol/L。经过14天的菌株培养与SPR检测(分析不同时间内的aflR基因的量),发现随着aflR基因在纯培养上的生成量,在第4-14天之间,并非与菌株的培养时间呈现一致性,而是随着菌株培养时间的延长而逐渐减少。
[Abstract]:Grain security is an important guarantee for people to maintain their basic life. As an important food crop, the safe storage of rice is the most important. Facilitate the adoption of effective measures to prevent or reduce pollution; To study the relationship between aflatoxin biosynthesis gene (aflR) and aflatoxin (aflatoxin) and to establish a method for rapid detection of aflR gene by SPR. It is expected that aflR gene, a precursor of aflatoxin synthesis, can be detected before aflatoxin is synthesized by microorganisms. In order to eliminate the threat of aflatoxin to people's health and reduce economic loss through rice, this paper takes the storage rice as the experimental material and adopts the classical microbiological method to separate and purify the aflatoxin. A strain producing aflatoxin was screened and identified by means of ELISA and PCR. Using RT-PCR method to clone aflR gene and SPR biosensor to detect nucleotide directly, a method for rapid detection of aflR gene by SPR was established, and the following conclusions were obtained: 1. Using 13 samples of rice stored in Yueyang and Pingtang areas as experimental materials, the number of fungi in rice was in the order of 105-106, which was a high level of mold pollution. And from the morphological analysis, the contaminated fungi mainly belong to Aspergillus and Penicillium. Aflatoxin was analyzed by ELISA method. Two strains producing aflatoxin were found, which were identified by morphological and its rRNA sequence analysis. The strain was identified as Aspergillus flavusus, named D1. 2. The aflR gene related to aflatoxin production was cloned by RT-PCR using A. flavus D1 as the experimental strain. 3. By using the direct method of DNA and DNA hybridization, a method for rapid detection of aflR gene by SPR was established. The lowest concentration of the standard probe was 7.5 nmol / L. The strain was cultured for 14 days and detected by SPR (to analyze the amount of aflR gene at different time). It was found that the amount of aflR gene produced in pure culture was not consistent with the culture time of the strain during the 4-14 days, but decreased gradually with the extension of the culture time of the strain.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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