The Research Of PCR Detedtion Method Of The Penicillum Expansum

Penicillum Expansum,which belongs to the fungi and decays apples, is patulin'smain producer. Patulin is a mycotoxin that that could cause teratogenic andcancerogenic. The concentration of patulin in food, especially in the juice is clearlyrequired. Therefore, it is essential to control the growth of the Penicillium Expansumto control the growth of patulin. The direct controlling method is to detect the mold.Traditional methods of identifying Penicillum Expansum were slow and inconclusiveand can be easily influenced by the subjective factors. So alternative approaches areneeded. Recent years have seen the fast development and application of PCR indetecting microorganism, and the fast and accurate characteristics have made itproperous in this area. However, the extracting method of the genomic DNAdetermins the discrimination of PCR. Therefore, it has significant role on improvingthe quality of the apple juice, on reducing the fungus which can produce themycotoxin.This study takes the fungus Penicillum Expansum, which can produce patulin, asthe main material. By comparing the five commonly used methods of extracting DNAfrom fungi, the most suitable extracting method for the genomic DNA of PenicillumExpansum was found. Then the specific primers which were suitable for this funguswere designed, and the conditions of the amplification reaction were optimized. Thisstudy provides the basic theory and research for practical application anddevelopment in the future.The main conclusions of this study are listed as follows:(1)Compared with the five common methods which were used to extract thegenomic DNA of Penicillum Expansum, the extractive effect of these methods, andwhether the DNA were suitable for the following research, we found that the benzylchloride method was the best to extract DNA from Penicillum Expansum, and it issatisfactory for the follow-up research.(2)Through optimizing the PCR conditions, the optimum PCR amplificationsystem was got as follow: Annealing temperature was57℃, the primers' concentration was0.40μmol/Land the mass concentration of template DNA was8μg/mL.(3)Through the specificity and sensitivity tests, we found that the reaction systemcould amplify the genomic of Penicillum Expansum specifically, while the length was288bp, and the limit of detecting DNA was0.1μg/mL. The results of the cloning andsequencing showed that the homology between the purposed gene fragment and thepart of the polygalacturonase sequence of Penicillum Expansum reached94%.The results of this research showed that this method could rapidly, specificallydetect Penicillum Expansum, and it could provide an effective theoretical guidance forcontrolling the growth of this mold, so as to prevent contamination of patulin. Andthis method could be used for business detection of the contamination of PenicillumExpansum in apple and juice.