Rapid Detection And Optimization Of Real Time PCR Of Patulin-Producing Penicillium Expansum

Polymerase chain reaction( PCR) is an elegent technique of creating copies of specific fragments of DNA, which rapidly amplifies a single DNA fragment into millions. The technology of PCR can be used to detect microorganisms quickly as long as the primers used are suitable.The purpose of this test is to study fast and specific detective method of patulin-producing Penicillium expansum and the optimization of parameters used in real time PCR detection of Penicillium expansum also was studied in this paper.Through pure culture of Penicillium expansum in malt extract agar and malt extract broth, and cultivating Penicillium expansum in apples, apple flesh and apple juice, the Penicillium expansum DNA was extracted through modified Cenis method. Two pairs of primers based on isoepoxydon dehydrogenase and polygalacturonase were used to amplify Penicillium expansum DNA, a method to specifically and quickly detect Penicillium expansum DNA was established ; the effect of annealing temperature, primer concentration and template concentration on the amplification were also studied to obtain the best parameters.The results of the studies are listed as follows:1,the result has showed that primersⅡ(POL)based on polygatacturonase have high specificity that only target DNA was amplified, however primersⅠ(ISO)based on isoepoxydon dehydrogenase have poor specificity. 5×10-6μg DNA per reaction in pure culture can be detected. The detection time (about 5h) is remarkably shortened compared with conventional plate culture method(about 4～6d);2,DNA from apples, apple flesh and apple juice cultured with Penicillium expansum was also remarkably amplified with high specificity, and without interference from apple DNA;3,Using lightcycler to conduct real time PCR, the best annealing temperature is 61℃,primer concentration is between 0.20μmol/L～0.40μmol/L,and template concentration had little effect on real time PCR. Using the above-mentioned parameters, the good PCR amplification curve, product melting curve and clear bands on electrophoresis were got.The PCR method established in this study is specific, fast and sensitive to detect Penicillium expansum, it can be used in detection of Penicillium expansum in apple juice production and quality control, it can also be used for tracing Penicillium expansum contamination in processing.The parameters to detect Penicillium expansum obtained in this study can be a reference for real time PCR detection for other microbes, it can be also used in the detection of Penicillium expansum in apple juice production and quality control.