本文關(guān)鍵詞：稻谷中霉菌分析及aflR基因的SPR快速檢測技術(shù)建立　出處：《湖南農(nóng)業(yè)大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 黃曲霉毒素 稻谷 aflR基因 SPR

【摘要】：糧食安全是人們維持基本生活的重要保障,稻谷作為人們生活重要的糧食作物,其安全貯藏是重中之重。研究分析倉儲稻谷中霉菌污染的狀況,便于采取有效的措施來防止或降低污染；研究污染稻谷中的黃曲霉毒素生物合成的關(guān)鍵調(diào)控基因aflR與黃曲霉毒素之間的關(guān)系并建立SPR快速檢測aflR基因的方法,有望在微生物未合成黃曲霉毒素前,檢測到合成黃曲霉毒素的前體物質(zhì)(aflR基因),起到預(yù)警的作用,杜絕黃曲霉毒素通過稻谷對人們身體健康的構(gòu)成威脅及減少經(jīng)濟損失。本文以倉儲稻谷為試驗材料,采用經(jīng)典微生物學(xué)分離純化方法,分析稻谷中主要污染霉菌類型,通過ELISA與PCR相結(jié)合的方法篩選并鑒定了一株產(chǎn)黃曲霉毒素的菌株；利用RT-PCR方法克隆aflR基因,以及采用SPR生物傳感器直接檢測核苷酸技術(shù),建立了SPR快速檢測aflR基因的方法,得到以下結(jié)論： 1、以來自岳陽、坪塘地區(qū)倉儲稻谷的13個樣本為試驗材料,分析稻谷中霉菌的數(shù)量均在105-106數(shù)量級之間,已經(jīng)屬于霉菌污染的較高水平；并且從形態(tài)學(xué)分析,污染的霉菌主要為曲霉屬和青霉屬。通過ELISA方法對污染頻率較高的菌株進行黃曲霉毒素分析,發(fā)現(xiàn)2株產(chǎn)黃曲霉毒素的菌株。以產(chǎn)毒較強的菌株作為本試驗的試驗菌株,利用形態(tài)學(xué)、ITS rRNA序列分析相結(jié)合的方法對其進行了鑒定,確認(rèn)該菌株為黃曲霉菌(Aspergillus flavus),命名為D1。 2、以A.flavus Dl為試驗菌株,通過RT-PCR方法,克隆了與黃曲霉毒素產(chǎn)生相關(guān)的調(diào)控基因aflR基因。 3、利用DNA與DNA雜交的直接法,建立了SPR快速檢測aflR基因的方法,檢測標(biāo)準(zhǔn)探針的最低濃度為7.5nmol/L。經(jīng)過14天的菌株培養(yǎng)與SPR檢測(分析不同時間內(nèi)的aflR基因的量),發(fā)現(xiàn)隨著aflR基因在純培養(yǎng)上的生成量,在第4-14天之間,并非與菌株的培養(yǎng)時間呈現(xiàn)一致性,而是隨著菌株培養(yǎng)時間的延長而逐漸減少。
[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.
【學(xué)位授予單位】：湖南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R155.5

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