Analysis Of Fungi In Paddy And Establishment Of Rapid Detection Method For AflR Gene Based On SPR Technology

Food security is necessary people’s daily life. Rice is an important food crop for it is one kind of staple food. Thus its storage plays important role. Studies and researches of mold contamination during rice storage has made the measurement, in order to prevent and/or reduce contamination, more effective. The relationship between aflatoxin and the key regulatory genes aflR of the aflatoxin biosynthesis in the contaminated rice was studied clearly and a SPR method for afIR gene rapid detection was established, making preliminary detection of aflatoxin by using its precursors (aflR genes) before it is synthesized by microorganism availabe. This result may be used as a preliminary warning and eliminate aflatoxin which threatens human health and causes economic losses.This research studied rice and analyzed the main mold contaminate by classical microorganism screening method. A strain producing aflatoxin was identified by the combination of ELISA and PCR method. RT-PCR and SPR biosensor were used to clone genes aflR and detect ribonucleotides respectively. At the end, a rapid method to detect afIR genes by SPR was established Different periods. The main conclusions from this study are:1.13rice samples from Yueyang and Pingtang storage houses were took as experimental materials. The amount of mold from each samples were between105-106, which is considered as a high degree of mold contamination. After morpya analysis, the contaminate molds were determined as Aspergillus and Penicillium. Aflatoxin synthesis capacity of the strains with more contamination frequency was analyzed; two strains produced aflatoxin. Strain D1had highest aflatoxin synthesis capacity and was identified as Aspergillus flavus through morphology and ITS rRNA sequences2. The A. flavus D1as experimental strain, regulatory genes afIR responsible related to aflatoxin synthesis with Aflatoxin produce from A. flavus D1was cloned by RT-PCR.3. DNA and DNA direct hybridization were used to establish the rapid method of afIR gene detection to detect genes afIR by SPR. The minimum concentration for standard probe detection was7.5nmol/L. Strain D1was cultured for14days and the amount of the afIR genes detected by SPR After culturing the strain D114d and SPR detection(to analyze the content of afIR gene in different time), the product of afIR gene were not increased, yet decreased during incubationdidn’t increase with the enlongation of culturing time, contrarily it decreased.