Traditional Chinese Medicinal Materials Surface Contamination Toxin-producing Fungus Rapid Detection Methods Of Research

AimsContamination of Chinese herbal drugs by mycotoxigenic fungi during preparation process and storage is of universal concern.Unwanted fungal growth not only results in reduction of active components of drugs,but also causes health risks for both humans and animals by producing various mycotoxins.Aim of this thesis was to develop a simple,sensitive and reliable multiplex-PCR-based method for rapid detection of potential aflatoxin-,sterigmatocystin-and ochratoxin-producing fungi in Chinese herbal drugs,establishing an early-warning system for mycotoxin contamination in Chinese herbal drugs.Methods1 Develop a multiplex-PCR(mPCR)method for the detection of mycotoxin(AFs,ST,OTA) producers based on pure strains2 Collect samples of TCM-drugs,modify the mPCR method for fungi eluted from the surface of TCM-drugs as well as DNA extraction protocol.3 Evaluate the sensibility and specificity of the above mPCR method with artificial inoculated TCM-drug(Ginseng was used as the representative.)4 Evaluate the applicability of the developed mPCR method to other TCM-drugs (Notoginseng radix et rhizome,Ilex asprella,Armeniacae semen amarum,Persicae semen,Dioscorea rhizoma,Dendrobii caulis,Rehmanniae radix praeparata,Morinda officinalis radix,Citri sarcodactylis fructus),by examining drug matrix effect on the sensibility of mPCR5 Analysis of fungal contamination in drug samples with the developed mPCR method6 Determine the concentration of mycotoxins(AFB1,AFB2,AFG1,AFG2,ST,OTA)in drug samples using LC/MS-MS.Compare the results with those of PCR methods7 Repeat the above experiments as described in e)with the same drug samples after an interval of time.Compare the results of the two experiments and those of PCR methods.Discuss the feasibility of the developed PCR methods in prognosis of fungal contamination in TCM-drugs.8 Evaluate the applicability of mPCR method to food samples(peanut,corn,wheat and sesame)and analysis of market samples ResultsTo establish mPCR detection methods,the primers used are extremely critical.After comparison of various primers targeting the key genes in mycotoxin biosynthesis,a multiple PCR method for detection of AFs-,ST-and OTA-producers based on pure strains was developed.Four primer pairs were used:AF-1 specific for AFs production,AFST-1 for AFs and ST,OTA-1 for OTA and the fungal universal primer Wen2.At the ratio of AF-1:OTA-1:AFST-1:wen2=2:2:1:2,the method is of good specificity and the sensitivity was determined as 10~3 spores/mL.DNA extraction method for fungi eluted from the surface of TCM-drugs was optimized,with Ginseng selected as the representative sample.The modified CTAB method was economic and more suitable for DNA extraction.Sensitivities of mPCR determined with addition of the extracts of Ginseng and other 9 TCM-drugs in the PCR reaction varied from10~3-10~4 spores/mL,which was comparable with pure strains.Except Shudi(Rehmannia glutinosa),sensitivities of mPCR determined with artificial inoculated drugs were all between 10~3-10~5 spores/mL,indicating that the mPCR method developed was robust and could be applied to detect the fungal contamination of most TCM-drugs.And then,the validated method was carried out to evaluate the contamination of AF-,ST-and OTA-producers in total 95 samples of 10 TCM-drugs collected from 4 local hospitals and 4 TCM pharmacies.AF-producers and OTA-producers were detected with contamination rate 8.4%and 9.5%,respectively.In contrast,ST-producers were most prevalent(18.9%)in the samples tested.95 samples of 10 TCM-drugs were also submitted to analysis of AFs,ST and OTA by LC-MS/MS.Only two samples were found to be contaminated with ST,which was consistent with the results of mPCR that ST-producers were detected in the corresponding samples.The results confirmed the reliability of the mPCR method in this study.Finally,the method was evaluated for the applicability to 4 food samples.The detection limits varied from 10~2-10~3 spores/mL,which was consistent with the results of the sensitivity test of pure strains.mPCR analysis of 22 samples of 4 kinds of foods randomly selected from 6 supermarkets in Guangzhou showed that 50%food samples were contaminated by fungi.Among them,ST-producers(13.6%)were the most contaminants,followed by AF-producers(4.5%).No OTA-producer were detected.ConclusionsIn this study,a simple,rapid,and stable method based on quadruple PCR technique was developed for direct detection of three categories of mycotoxigenic fungi(AF,ST and OTA)from the surface of TCM-drugs and foods.The method can directly detect the toxicgenic fungi without the traditional culture and purification of the sample surface bacteria.The method was applied to the detection of 10 kinds of Chinese herbal medicines and 4kinds of foods.The results showed that the contamination of ST producers was most serious.Implying special attention should be paid to evaluate the potential hazard caused by ST producers in TCM-drugs and foods.