Identification Of Fourteen Mycotoxigenic Fungi On The Surface Chinese Herbal Drugs By Using Multiplex PCR And Optimization Of The Storage Conditions

ObjectiveObjective of this thesis was to develop a sensitive,simple and reliable multiplex-PCR(mPCR)method for rapid detection of potential aflatoxin-,fumonisin-,sterigmatocystinand ochratoxin-producing fungi on the surface of Chinese herbal drugs(TCM-drugs)and foods,establishing an early-warning system for mycotoxin contamination in Chinese herbal drugs and foods.Additionally,the thesis aimed to optimize,the storage conditions(temperature and humidity)of TCM-drugs to minimize the contamination of sterigmatocystin.taking Ilex asprella as representative.MethodsA multiplex-PCR method for the detection of mycotoxin(AFs,FBs,ST,OTA)producers was firstly developed based on pure strains.The method was applied to analyze 15 TCM-drugs(Ilex asprella,Armeniacae semen amarum,Persicae semen,etc).The sensibility and specificity of the method were evaluated with the addition of drug matrix or artificial inoculated TCM-drug in PCR solution.Finally,the approach was applied to detect the fungal contamination of the samples of 15 TCM-drugs and foods collected from different markets.In order to test the reliability of the method,LC/MS-MS was used to determine the concentration of 8 mycotoxins such as FB1,FB2,AFB1,AFB2,AFG1,AFG2,ST,OTA in drug samples.At the same time,the plate dilution method was used to isolate,purify and culture the surface-contaminated fungi.The universal primer ITS 1/ITS4 was used to identify fungi.The strain species were determined and liquid culture was carried out to detect whether the strain produced mycotoxin.Experiments based on response surface methodology combined central composite design(RSM-CCD)were carried out to determine the optimal storage conditions of Ilex asprella for protection against the contamination of Aspergillus versicolor.The herb contaminated with Aspergillus versicolor was stored under different temperatures and humidity,to determine the conditions unsuitable for the growth of Aspergillus versicolor and ST accumulation and optimize the storage conditions of Ilex asprella.ResultsThe optimal primer combination for multiplex PCR consists of 4 pairs of specific primers and a ITS primer pair.When the molar ratio of 5 primer pairs was AF-1:AFST-1:OTA-1:wen2:Fum 13=2:1:2:2:2,the multiplex PCR method has good accuracy and specificity.The detection limit of the pure strain by multiplex PCR was 103 spores/mL.The sensitivity of multiplex PCR for simultaneous detection of four toxin-producing fungi containing TCMs was 103-104 spores/mL(except Lycium barbarumL).At the same time,artificial infection TCMs and foods were detected by multiplex PCR.The results showed that the detection limit was 103-104 spores/mL(except Lycium barbarumL).and no other bands appeared,indicating that the multiple PCR detection method has high accuracy.A total of 113 samples of 15 kinds of TCMs purchased from 7 pharmacies and 5 hospitals in Guangzhou were detected by multiplex PCR.The results showed that the contamination rate of fungi in TCMs was 40.7%.The most serious contamination was ST-producing fungi(18.6%)followed by AF-producing fungi(13.3%).LC-MS/MS was used to detect the above corresponding samples.The consistency between the detected toxins and the corresponding toxin-producing fungi was 89.2%.The accuracy rate of early warning by multiplex PCR was 95.4%.No warning effect was 4.6%.In order to further verify the reliability of the multiplex PCR method,traditional isolation and identification of fungi were used for 15 Chinese medicinal materials.It was found that the consistency of this method and multiplex PCR method was 87.6%,and the accuracy of early warning was 95%.At the same time,33 batches of food samples from 4 kinds of food in 4 supermarkets were detected by multiplex PCR.The results showed that the contamination rate of food fungi was 48.5%,and the most serious contamination was FBs-producing fungi,up to 24.2%.and comparing the two methods with four kinds of food,the accuracy of early warning was 87.5%.The herb contaminated with Aspergillus versicolor was stored under different temperatures(20℃-40℃)and humidity(80%-95%)for 7 days.In the study of the optimal storage conditions of Ilex asprella,when the temperature range was 26-34℃ and the humidity was 85-90%,a large amount of variegated toxin was produced.The finding suggested that Ilex asprella should be stored in an environment with humidity and temperature below 85%and 26℃ respectively,to reduce Aspergillus versicolor growth.ConclusionsThis study established a simple,rapid and stable PCR method for the direct detection of TCMs and foods surface contamination AFs,ST,OTA,FBs-producing fungus without isolation and purification.The results showed that fungal contamination was very common.While contamination of Aspergillus versicolor in the TCMs was the most serious,fumonisin-producing fungis in the foods were the most serious,which was consistent with the previous literature reports,and should raise and attention.The problem of toxin-producing fungi and mycotoxin contamination is an urgent problem in the storage process of TCMs.It is an efficient and easy way to control the growth and production of toxins by controlling the storage environment conditions.This study showed that A.versicolor growth and toxicity were affected by environmental temperature and humidity.The effects of humidity on growth,mycotoxin production and the alteration of effective components were higher than those of temperature.Low temperature and humidity could significantly reduce ST content while maintaining the amounts of active ingredients.This study showed that it was scientific and feasible to use this method to assess the conditions of herb storage,and provided new clues for controlling the pollution of medicinal plants by mycotoxin.