基于高通量测序法及平板法研究龟甲饮片表面真菌种类及其多样性
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摘要
目的检测龟甲饮片表面真菌种类及多样性,为龟甲饮片用药安全提供参考依据。方法从北京市零售药店收集龟甲饮片8批,采用高通量测序法对龟甲饮片表面真菌种类及真菌多样性进行预估。同时,采用平板法培养、纯化龟甲表面真菌,获得单一菌株,依据菌落形态、显微结构、DNA条形码对真菌进行鉴定。结果高通量测序法结果显示,龟甲饮片表面真菌共计2界5门21纲56目123科173属201种,主要为曲霉属(Aspergillus)、虫草属(Cordyceps)、酵母菌属(Wickerhamamyces)、枝孢霉属(Cladosporium)、链格孢属(Alternaria)、念珠菌属(Candida)、棒杆菌属(Wallemia)、青霉属(Penicillium)。平板法检测结果显示,龟甲饮片表面真菌共计42株10种,主要为草酸青霉菌(Penicillium oxalicum)、烟曲霉(Aspergillus fumigates)、黑曲霉(Asper-gillus niger)、阿曲霉(Aspergillus amstelodami)、青霉属(Penicilliumsp.)、总状毛霉变种(Mucorracemosus f.racemosus)、白曲菌(Aspergillus candidus)、散囊菌属(Eurotiumsp.)。结论平板法可对中药饮片表面真菌进行分离纯化,高通量测序法则快速高效,综合平板法与高通量测序法可以科学、准确、全面地鉴定龟甲表面的真菌种类,为龟甲饮片的安全使用提供检测方法。
Objective To detect the species and diversity of fungi on surface of tortoiseshell decocting pieces and provide reference for medication safety of tortoiseshell decocting pieces.Methods The decocting pieces of tortoiseshell(8 batches)were collected from retail pharmacies in Beijing,and species and diversity of fungi on surface of tortoiseshell decocting pieces were estimated by using method of high-throughput sequencing(HTS).At the same time,fungi on surface of tortoiseshell decocting pieces were cultured by using plate method,and then were purified by using top purification method for obtaining a single fungus strain.The fungi on the surface of tortoiseshell decocting pieces were identified according to colony morphology,microstructure,and DNA barcodes.Results The results of HTS showed that there were 2 lines,5 doors,21 classes,56 orders,123 families,173 genera and 201 species of fungi on surface of tortoiseshell decocting pieces summed up totally,including mainly Aspergillus,Cordyceps,Wickerhamamyces,Cladosporium,Alternaria,Candida,Wallemia and Penicillium.The results of plate method showed that there were 42 strains of 10 species of fungi on surface of tortoiseshell decocting pieces summed up totally,including mainly Penicillium oxalicum,Aspergillus fumigates,Aspergillus niger,Aspergillus amstelodami,Penicillium sp.,Mucor racemosus f.racemosus,Aspergillus candidus and Eurotium sp.Conclusion The fungi on surface of tortoiseshell decocting pieces can be separated and purified by using plate method.HTS is rapid and efficient.The combination of plate method and HTS can identify scientifically,accurately and comprehensively the species of fungi on surface of tortoiseshell decocting pieces,and provide a detection method for medication safety of tortoiseshell decocting pieces.
Objective To detect the species and diversity of fungi on surface of tortoiseshell decocting pieces and provide reference for medication safety of tortoiseshell decocting pieces.Methods The decocting pieces of tortoiseshell(8 batches)were collected from retail pharmacies in Beijing,and species and diversity of fungi on surface of tortoiseshell decocting pieces were estimated by using method of high-throughput sequencing(HTS).At the same time,fungi on surface of tortoiseshell decocting pieces were cultured by using plate method,and then were purified by using top purification method for obtaining a single fungus strain.The fungi on the surface of tortoiseshell decocting pieces were identified according to colony morphology,microstructure,and DNA barcodes.Results The results of HTS showed that there were 2 lines,5 doors,21 classes,56 orders,123 families,173 genera and 201 species of fungi on surface of tortoiseshell decocting pieces summed up totally,including mainly Aspergillus,Cordyceps,Wickerhamamyces,Cladosporium,Alternaria,Candida,Wallemia and Penicillium.The results of plate method showed that there were 42 strains of 10 species of fungi on surface of tortoiseshell decocting pieces summed up totally,including mainly Penicillium oxalicum,Aspergillus fumigates,Aspergillus niger,Aspergillus amstelodami,Penicillium sp.,Mucor racemosus f.racemosus,Aspergillus candidus and Eurotium sp.Conclusion The fungi on surface of tortoiseshell decocting pieces can be separated and purified by using plate method.HTS is rapid and efficient.The combination of plate method and HTS can identify scientifically,accurately and comprehensively the species of fungi on surface of tortoiseshell decocting pieces,and provide a detection method for medication safety of tortoiseshell decocting pieces.
引文
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[2]王文丽.15种中药材表面污染真菌的分离鉴定及产毒性能分析[D].广州:广州中医药大学,2013.Wang WL.Analysis of the separation and identification of fungal contamination and their mycotoxin-producing potential on the surface of fifteen traditional herbal medicines[D].Guangzhou:Guangzhou University of Chinese Medicine,2013.
[3]秦筱茂.九种药材中真菌种类及污染真菌毒素初探[D].北京:北京协和医学院,2011.Qin XM.Studies on fungal species and related mycotoxin contamination in 9 Chinese medicinal herbs[D].Beijing:Peking Union Medical College,2011.
[4]李闽真,马群飞,傅武胜.市售中药材霉菌污染情况和优势种群分布的研究[J].中国卫生检验杂志,2013,23(17):3351-3353.Li MZ,Ma QF,Fu WS.Research on dominant species distribution and pollution status of molds in commercial Chinese herbal medicine[J].Chin J Health Lab Tec,2013,23(17):3351-3353.
[5]王新雨,谭晓梅,陈飞龙,等.HPLC-ELSD法测定醋龟甲中羟脯氨酸[J].中草药,2011,42(7):1338-1340.Wang XY,Tan XM,Chen FL,et al.Determination of hydroxyproline in vinegar Testudinis Carapax et Plastrum by HPLC-ELSD[J].Chinese Traditional and Herbal Drugs,2011,42(07):1338-1340.
[6]方文忠,葛尔宁,盛振华.鳖甲、龟甲中脂肪酸的GC-MS比较分析[J].内蒙古中医药,2013,32(17):43-45.Fang WZ,Ge EN,Sheng ZH.The Analysis of fatty acids in Carapax Trionycis and Carapax et Plastrum Testudinis by GC-MS[J].Nei Mongol Journal of Traditional Chinese Medicine,2013,32(17):43-45.
[7]徐清,李梦,张帆,等.采用电感耦合等离子质谱法检测龟甲及龟甲胶药材中重金属元素的含量[J].北京中医药大学学报,2018,41(8):670-674.Xu Q,Li M,Zhang F,et al.Detection of the content of heavy metal elements in tortoise shell and tortoise shell glue by using inductively coupled plasma mass spectrometry[J].Journal of Beijing University of Traditional Chinese Medicine,2018,41(8):670-674.
[8]刘英华,陈瑛.高通量测序技术的最新研究进展[J].中国妇幼保健,2013,28(12):1990-1992.Liu YH,Chen Y.The latest research progress of highthroughput sequencing technology[J].Maternal and Child Health Care of China,2013,28(12):1990-1992.
[9]乌日拉嘎,徐海燕,冯淑贞,等.测序技术的研究进展及三代测序的应用[J].中国乳品工业,2016,44(4):33-37.Wurilaga,Xu HY,Feng SZ,et al.Research progress of sequencing technologies and the application of third generation sequencing[J].China Dairy Industry,2016,44(4):33-37.
[10]王兴春,杨致荣,王敏,等.高通量测序技术及其应用[J].中国生物工程杂志,2012,32(1):109-114.Wang XC,Yang ZR,Wang M,et al.High-throughput sequencing technology and its application[J].China Biotechnology,2012,32(1):109-114.
[11]Grice EA,Kong HH,Conlan S,et al.Topographical and temporal diversity of the human skin microbiome[J].Science,2009,324(5931):1190-1192.
[12]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979:499.Wei JC.Fungal Identification Manual[M].Shanghai:Shanghai Scientific and Technical Publishers,1979:499.
[13]Johnston PR,Park D,Smissen RD.Comparing diversity of fungi from living leaves using culturing and high-throughput environmental sequencing[J].Mycologia,2017,109(1):1-12.
[14]苏锡南.环境微生物学[M].北京:中国环境科学出版社,2015:4-5.Su XN.Environmental Microbiology[M].Beijing:China Environmental Science Press,2015:4-5.
[15]颜仁梁.中药储存与养护[M].重庆:重庆大学出版社,2014:12-13.Yan RL.Storage and Conservation of Chinese Medicinal[M].Chongqing:Chongqing University Press,2014:12-13.