DNA提取过程中混入的实验室空气微生物DNA定量

Quantification of microbial DNA in laboratory environment during DNA extraction

摘要:

元基因组测序方法为微生物研究提供了有力的工具。但其中的DNA提取过程，会不可避免地混入实验室中的空气微生物。这些微生物DNA，是否会对一些极微量的元基因组检测 (如皮肤样本等) 结果造成影响，有多大影响，仍没有明确结论。本研究首先收集了实验室空气样品，用16S rRNA引物建立了基于qPCR的标准曲线，并检测了在开放环境下提取DNA过程中可掺杂的环境微生物DNA量。然后在开放环境下提取纯水DNA样品并进行元基因组分析，以确定掺杂环境微生物的种类。最后分别在生物安全柜和实验室开放环境下提取皮肤样本，并用鸟枪测序方法对样本的微生物组成进行分析，以评估掺杂环境微生物对元基因组检测结果的影响。结果显示，在实验室开放环境的DNA提取过程中，环境微生物的DNA残留可达28.9 pg，可达某些极微量样本DNA总量的30%。元基因组分析显示，样品中掺杂的环境微生物主要是痤疮杆菌Cutibacterium acnes、大肠杆菌Escherichia coli等皮肤常见细菌。与洁净皮肤样本的信息相比，开放环境下提取掺杂了数十种环境微生物，并导致主要菌种的丰度大幅降低，从而影响结果的真实性。因此，微量样品的DNA提取应在洁净环境下执行。

Abstract:

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.