地龙饮片表面真菌的多样性研究

Diversity of fungi on surface of Pheretima

中药材在种植、采收、加工、运输及贮藏过程中容易受到真菌污染,2015年版《中国药典》规定检测地龙饮片的黄曲霉毒素,经查阅文献发现国内外目前尚未有学者对地龙饮片的表面真菌进行研究。该实验从北京市零售药店收集地龙饮片8批,采用传统的平板法培养地龙饮片表面的真菌,顶端纯化法获得单一菌株,通过真菌的菌落形态、显微结构特征、DNA条形码3种方法鉴定真菌。同时,基于Illumina Hi Seq 2500高通量测序平台,分析地龙饮片表面真菌的多样性。结果显示,平板法共分离鉴定得到287株9种真菌,结合3种真菌鉴定方法,9种真菌中的8种可以鉴定,分别为黑曲霉菌、青霉菌、链格孢菌、黄曲霉菌、草酸青霉菌、腐质霉菌、Talaromyces purpurogenus和Aspergillus insuetus,1种真菌未得到结果。其中,青霉属、曲霉属真菌为主要污染菌。高通量测序结果于不同分类水平分属2个界、6个门、19个纲、44个目、98个科、127个属、121个种,优势真菌属为节担菌属、曲霉属和虫草属,相对丰度分别为63. 33%,15. 28%,10. 28%。通过对北京市零售药店的地龙饮片表面真菌进行多样性研究,可以为其安全贮藏和临床用药安全提供一定的参考依据。

Traditional Chinese medicines(TCMs)are easily contaminated by fungi during planting,harvesting,processing,transportation and storage.The 2015 version of Chinese Pharmacopoeia stipulates the detection of aflatoxin in Dilong.After reviewing the literature,it has been found that there are no domestic and foreign scholars who have studied the surface fungi of Dilong.Pheretima,known as Dilong in China,is a commonly used TCMs in animal.In this experiment,8 batches of Dilong were collected from retail pharmacies in Beijing.The fungi on the surface of Dilong were cultured by traditional plate method and the single strain was obtained by the top purification method.The fungal colony morphology,microstructure characteristics and DNA barcode were used to isolate and identify the fungi.At the same time,based on Illumina Hi Seq 2500 high-throughput sequencing platform,the diversity of fungi on the surface of Dilong was analyzed.The results showed that 287 strains of 9 species of fungi were isolated and identified by plate method.Combined with 3 kinds of identification method,eight of nine fungi could be identified,respectively,Aspergillus niger,Penicillium,Alternaria nees,A.flavus,and Penicillium oxalicum,Humicola sp.,Talaromyces purpurogenus and A.insuetus,1 kind of fungi was not identified yet.Among them,Penicillium and Aspergillus were the dominant genus.The results of high-throughput sequencing belonged to 2 boundaries,6 gates,19 classes,44 orders,98 families,127 genus and 121 species in different classification levels.Wallemia,Aspergillus and Cordyceps were the dominant genus,and the relative abundances are 63.33%,15.28%,and 10.28%,respectively.Through the diversity study on the surface fungi of Dilong in Beijing retail pharmacies,it can provide a reference for its safe storage and clinical use.