多种测序技术在药品检测环境微生物鉴定分析中的应用研究

目的:探索多种测序技术在药品检测环境微生物鉴定分析中的应用。方法:从药品微生物实验室环境中连续7个月采样收集获得248株细菌和6株霉菌,并对收集的细菌采用全自动微生物生化鉴定仪(Vitek 2 Compact)进行鉴定。根据生化鉴定结果选择在种属分类学上具有代表性的20种细菌(共28株)和6株霉菌分别采用一代测序技术(ABI 3500测序平台)和宏基因组测序技术(Hiseq 2000测序平台和Ion torrent测序平台)进行鉴定分析,相互验证菌株鉴定方法的准确性。结果:28株细菌的16S rDNA一代测序鉴定与生化鉴定相比,属水平分类一致的有23株,种水平分类一致的有10株。采用Hiseq 2000对28株细菌混合进行全基因组测序鉴定获得78种菌株信息,与16S rDNA一代测序相比,种水平一致的有15株,属水平一致的有2株,缺失了3株菌的信息,多出了61株菌的信息;霉菌全基因组测序获得3株菌信息,与霉菌LSU rDNA的一代测序结果相比,缺失了3株菌的信息。采用Ion torrent对28株菌混合进行16SrDNA宏基因组测序鉴定,获得信息包含7科、10属和13种,与16S rDNA一代测序结果对应的有11种,且10种属覆盖了一代测序中的28株菌;霉菌ITS2宏基因组测序结果包含4属和3种, 4属覆盖了霉菌一代测序结果中的6株菌,但种水平只有烟曲霉和一代测序结果一致。结论:新一代宏基因组测序技术及分析方法需要进一步改进与完善,才能对环境微生物混合菌株进行快速准确鉴定从而开展建库溯源工作。

Objective:To explore the application of a variety of sequencing technology in the identification of environmental microorganisms in medicines. Methods:Totally 248 strains of bacteria and 6 strains of fungi were collected from the author's pharmaceutical microbiology laboratory during consecutive 7 months, and these microorganisms were identified by biochemical identification instrument of Vitek 2 Compact. The representative 20 kinds of bacteria(28 strains)and 6 strains of fungi based on species taxonomy were chosen and identified by first-generation sequencing technology(ABI 3500 sequencer)and metagenomic pyrosequencing(Hiseq 2000 and Ion torrent)respectively to mutually validate the accuracy of strain identification method. Results:The comparison between the identification by 16S rDNA first-generation sequencing and the biochemical identification of 28 strains of bacteria showed that 23 isolates were at a consistent species level and 10 isolates at a consistent genus level.The whole genome sequencing of 28 mixed strains of bacteria were identified by Hiseq 2000, which provided information of 78 species of bacteria. Compared with 16S rDNA first-generation sequencing, 15 isolates at a consistent species level and 2 isolates at a consistent genus level were obtained, 3 isolates were missed and 61 isolates were obtained additionally; 3 isolates were obtained by the whole genome information sequencing and 3 isolates were missed when compared with the result by fungi LSU rDNA firstgeneration of sequencing. The 28 strains of bacteria by Ion torrent were mixed and identified by 16S rDNA metagenomic sequencing, and the information of 7 families, 10 genera and 13 species were obtained. 11 isolates were at a consistent species level and 10 genera covered 28 isolates of first-generation sequencing compared with 16S rDNA first-generation sequencing. Totally 4 genera and 3 species were obtained by metagenomic sequencing of fungi ITS2; 4 genera covered 6 isolates of first-generation fungus sequencing, but only Aspergillus fumigatus was consistent with the first-generation sequencing results on species level. Conclusion:Metagenomic pyrosequencing and analysis methods need further improvement and perfection to accurately identify the mixed strains of environmental microbes rapidly and carry out the construction and traceability of microbe database.