【作者】 何鸿举；

【导师】 樊明涛；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2011， 硕士

【摘要】 扩展青霉(Penicillium expansum)是一种多细胞丝状真菌,是棒曲霉素的主要产生菌,棒曲霉素是一种致癌、致畸毒素。传统的扩展青霉检测主要是培养法,耗时长,无法实现快速检测的目的,近年来PCR技术在微生物的检测方面具有明显优势,因此,研究如何检测棒曲霉素产生菌-扩展青霉,并设法在工艺过程中对其进行控制,减少该棒曲霉素产生菌,对提高苹果汁质量与安全具有重大意义。本试验以分离自腐烂苹果中的各真菌和标准扩展青霉菌株为试验原材料,基于PCR检测特异性的优点,根据扩展青霉polygalacturonase基因内一段保守序列设计了特异性PCR扩增引物,优化了PCR扩增条件,获得了一套最适扩增扩展青霉DNA的优化参数,以期为此技术的实际应用与推广提供理论依据和方法学参考。本试验主要获得了一下结果:(1)以标准扩展青霉菌为试验材料,筛选获得了最适合提取扩展青霉菌基因组DNA的方法—玻璃珠+氯化苄法。其他方法相对本试验而言,均不能满足要求;(2)以扩展青霉菌的polygalacturonase基因序列为靶目标,设计合成高特异性的PCR扩增引物,对扩展青霉菌实施PCR扩增,菌体的检测限可达1.34×103个孢子/mL,DNA的检测限可达2.40×10-2μg/mL;(3)对PCR技术的检测条件进行了一系列优化试验,获得了一套最适合检测扩展青霉菌的PCR扩增体系。同时对分离真菌和扩展青霉菌同时进行PCR扩增,验证了PCR检测的高特异性和高灵敏性。本研究结果为前期控制苹果中扩展青霉菌的产生、防止棒曲霉素的污染提供了有效的理论指导,可用于商业苹果感染扩展青霉菌的检测,亦可作为使用此技术检测其他微生物的方法学参考。更多还原

【Abstract】 Penicillium expansum is a kind of multicellular filamentous fungi, it is mainly producing patulin which is one of carcinogenic and teratogenic toxin. The traditional method of detecting Penicillium expansum by cultivating that is time-consuming and poor efficient, however using emerging PCR technology for detecting microbe has obvious advantages in recent years. Therefore, it is great significance by means of PCR technology to detect and decrease patulin-producing fungi-Penicillium expansum, thereby controlling process and improving apple juices quality. This experiment used various fungi strains isolated from rotten apples and standard Penicillium expansum strain as raw experiment materials, at the same time, designed a pair of specific PCR primers based on a conservative sequence of polygalacturonase gene of Penicillium expansum and optimized the PCR conditions to obtain a set of optimal PCR amplification parameters and provide the theory basis and the methodology reference. This experiment mainly obtained the results as follows:(1) Screened and obtained the optimal method for extracting Penicillium expansum DNA from the standard Penicillium expansum strain, glass beads with benzyl chloride method. Other methods were not suitable for the experiment.(2) Designed and synthetized high specific PCR primers to amplify Penicillium expansum DNA based on the polygalacturonase gene of Penicillium expansum. As a result, the limit of detecting fungi and Penicillium expansum DNA were 1.34 x 103 spores/mL and 2.40×10-2μg/mL respectively.(3) Optimized the PCR conditions and obtained a set of optimal parameters for amplifying Penicillium expansum DNA, meanwhile, tested and verified the high specificity and sensitivity of PCR detection.The results of this study provide effective theory guidance for controlling emerge of Penicillium expansum and preventing patulin contamination, which can be applied to detect Penicillium expansum and other microbes.更多还原