| 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. |
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