Screening Of Various Toxic Fungi From Traditional Chinese Medicine Based On MLPA And LC/MS~2 Method

Objective:To establish a method for simultaneously detecting the contamination of multiple toxigenic fungal and solving the problem of immediate prevention and control of fungal contamination in Chinese medicinal materials,which based on a rapid screening technique for molecular diagnosis combining modern chromatographic analysis techniques,we explored the biosynthetic pathway genes of mycotoxin and the characteristics of related synthetic genes,and designed the specific probes for toxin synthesis pathway according to the sequence conservativeness and variability of these genes.Method:The multiple connected probe amplification(MLPA)was employed for its advantages of the technology efficient,specific and sensitive,and the MLPA probes of their key genes on the toxin synthesis were designed,respectively,using Aspergillus flavus,A.ochraceus and Fusarium tricinctum as the research object,after MLPA experimental hybrid and amplification,capillary electrophoresis was used to detect the results.For the complex of Chinese medicinal materials matrix,which is often accompanied by the simultaneous pollution of a variety of fungi,produces a variety of mycotoxins.In the present study,the animal medicinal material Eupolyphaga Steleophaga was taken as an example.A method for detecting 12 mycotoxins in medicinal materials by LC-MS~2was established,where the acetonitrile-5mmol ammonium acetate(pH=3.0)was used as mobile phase,and gradient elution,multi-reaction monitoring and positive and negative ion scanning were also selected.The above two methods were established to detect the medicinal materials,with 8.5%sterile sodium chloride solution was added to the samples,and the fungi were released by shock.Then the fungal DNA was extracted using a fungal genomic DNA extraction kit,and the MLPA technique was used to detect the contamination of aspergillus flavus,aspergillus ochratreum and fusarium trilinear.Meanwhile,the content of the mycotoxins mentioned above in medicinal materials were determined by LC-MS~2 method.In addition,the content of aflatoxin in crude drugs was also determined referring to the methods in Chinese pharmacopoeia2015.Finally,the results of these detection methods were compared to further analyze the relationship between fungi and toxin production.Results:The specificity test of the MLPA method showed that the probes of each strain are specific and can only identify the respective target fragments,and cannot amplify other fungi,and no cross reaction was observed between the probes.Sensitivity test results indicate that the minimum detection limit for 3-fold MLPA amplification can reach 200copies of a single fungal nucleic acid.The content of the 12 mycotoxins in Chinese medicinal materials was detected by LC-MS~2 method.The established method showed good stability,and the results of standard recovery were all within the acceptable range.The RSD value was1.5%~13%,which can be effectively used for the simultaneous detection of a variety of mycotoxins in Chinese medicinal materials.From the market sample verification,MLPA method can be effectively used for the detection of various Chinese medicinal materials such as peach kernel,almond,terrapin,cardamom and so on.The possibility of medicinal herbs contaminating a single strain was low.Comparative analysis of the three detection results showed that MLPA method had a higher detection sensitivity,and the number of fungi within a certain range produced fewer toxins or were not detected.The more the number of fungi outside the range,the higher the toxin content.Conclusion:The MLPA detection method established in this study can achieve in one sampling,one analysis,and the purpose of detecting three kinds of fungi at the same time.This technology is highly specific,sensitive,and the advantages of its multiplicity detection,it plays an important role in the detection of fungi in Chinese herbal medicines and the prevention of toxin production.The combination of the two methods established provide a rapid screening approach for the contamination of Chinese herbal medicines.the detection method established in this study can provide technical support and theoretical basis for the detection and content determination of mycotoxin contamination of Chinese medicinal materials.