A New Technology For The Detection Of Aspergillus Flavus And Its Toxins In Chinese Herbal Medicines

1.Research SignificanceControl of the aflatoxin contamination is the core for the safety of Chinese herbal medicines.However,aflatoxin contamination might pose a threat to the health of users.Aspergillus flavus is a kind of multicellular microorganism belong to Aspergillus,which is widely spread in air,water,soil and natural environment and is the main contamination of Chinese herbal medicines.A large number of aflatoxin will be produced in appropriate conditions when Chinese herbal medicines are contaminated by Aspergillus flavus,while aflatoxin is difficult to eliminate.Therefore,the "early warning monitoring" of Aspergillus flavus is a key in the control of aflatoxin contamination and cross contamination of Chinese herbal medicines.Aflatoxins(Aflatoxins,AFT)are classified as Class I carcinogens by the World Health Organization,which have serious damage on human and animal liver tissue and lead to liver cancer,chronic hepatitis,jaundice hepatitis,hepatomegaly and cirrhosis,especially the toxicity and carcinogenicity of aflatoxin B1 is the most common.Recent years,with the wide application of Chinese herbal medicines,researchers found the phenomenon of excessive contamination of aflatoxins in the tangerine peel,almonds,astragalus,earthworm etc.,which may be a serious threat to the use of security.2.Research ProgressTraditional morphological methods is generally used in the identification and classification of fungi,while this requires researchers have professional knowledge and have the drawbacs of inaccurate identification,time-consuming etc.However,the molecular detection method has been studied and widely applied in the detection and identification of toxic fungi because of its high accuracy,short time-consuming and strong objectivity.The current molecular methods to detect Aspergillus flavus include DNA sequencing,PCR-restriction fragment length polymorphism,multiplex PCR,fluorogenic quantitative PCR etc.However,those detection methods are time-consuming,generally takes more than 2 hours,especially DNA sequencing will take longer,which can’t meet the need of rapid qualitative and quantitative detection for large samples.Therefore,there is an urgent need to develop a more convenient and accurate qualitative and quantitative methods for the current Aspergillus flavus contamination detection.At present,the widely applied aflatoxin detection technology mainly includes instrumental analysis method and immunological analysis method.Instrument analysis methods mainly involve thin layer method,liquid chromatography,gas chromatography and liquid chromatography-mass spectrometry,etc.Immunological analysis methods mainly include enzyme-linked immunoassay and lateral flow tomography detection technology and its products.However,the instrumental analysis method requires expensive instruments,skilled technicians,and complex sample pretreatment,which is not suitable for rapid detection of large number samples.Although immunoassay methods take less time,it still spends more than 2 hours,and the cost is also high.Therefore,there is urgent need to develop a faster,more efficient and less cost technique for the detection of aflatoxin.3.Research Objective(1)Establish loop-mediated isothermal amplification technique to rapidly detect the toxin Aspergillus flavus in Chinese herbal medicines;(2)Establish fluorescence polarization immunoassay technique to rapid qualitative and quantitative of aflatoxin B1 in Chinese herbal medicines;(3)Synchronous high-throughput qualitative and quantitative detection of toxin Aspergillus flavus and aflatoxin.4.Research and Results(1)The culture and identification of Aspergillus flavus and other fungi in Chinese herb medicines.The Aspergillus flavus was cultured by selective cultivation and other fungi were cultured in PDA culture medium,then identified by ITS bar code and morphological identification method.(2)Established a loop-mediated isothermal detection method.Designed the amplification primers of ring-directed isothermal amplification system based on the exon sequence of functional gene VER-1,and optimized the system for stable and efficient amplification,then combined real-time fluorescence detection to develop real-time fluorescent loop mediated isothermal amplification technique for the detection of Aspergillus flavus.(3)Established a quantitative PCR detection method.In this study,specific primers were designed for the biosynthesis of key genes in aflatoxin,and qualitative and quantitative detection of Aspergillus flavus was carried out by real-time fluorescence quantitative PCR.(4)Established a fluorescence polarization immunoassay detection method.In this study,the aflatoxin was combined with amino fluorescein by chemical synthesis to obtain a fluorescent label capable of binding to aflatoxin antibody and having a fluorescent signal.The synthesized products were identified by thin layer chromatography and mass spectrometry.Because of the competition between aflatoxin and fluorescent labeled aflatoxin to aflatoxin antibody,the molecular weight and rotation speed of the aflatoxin and aflatoxin fluorescent protein changed,and caused the change of fluorescence polarization value,which leaded to the acquirement of linear regression curve and realized the qualitative and quantitative detection of aflatoxin.(5)Established a HPLC detection method of aflatoxin.In this study,the derivatives of aflatoxin standard were derivatized to obtain a stronger and more stable derivatized product of fluorescence signal,and qualitative and quantitative analysis was carried out by fluorescence detector coupled with high performance liquid chromatography.(6)Finished the collection of totally 72 samples from four different parts of the six representative Chinese herb medicines,and detection of Aspergillus flavus by loop-mediated isothermal amplification and fluorescent quantitative PCR,and detection of aflatoxin by fluorescence polarization and high performance liquid chromatography.