叠氮溴化丙锭筛选环境活性微生物的原理及其应用

Principle of propidium monoazide in screening viable microorganisms from environmental samples and its application

环境微生物总DNA主要来源于游离DNA以及活性和非活性微生物群。因此,单纯基于环境微生物总DNA的直接研究技术可能无法反映出环境中真实的活性微生物群落结构及组成,极易造成微生物丰度及群落多样性的高估,急需建立有效可行的活性微生物筛选方法。叠氮溴化丙锭(Propidium monoazide,PMA)是一种能够不可逆地共价结合死细胞DNA和胞外DNA(统称为relic DNA)的光敏性核酸染料,致使relic DNA被钝化,不能完成PCR扩增。基于此,结合后续荧光定量PCR和高通量测序等分子生物学技术,可快速、准确且高效地筛选区分出环境样本中的活性微生物类群。本文针对近几年关于PMA染料法在底泥、土壤和水体等常见环境样品中的应用研究结果进行综述,并探讨了目前PMA染料法应用中存在的问题,提出了相应的改进和完善对策,为深入研究复杂环境样品活性微生物类群及其结构功能提供了新思路。

Microbial total DNA obtained from environmental samples comes from cell-free DNA,intracellular DNA of viable and nonviable microbes.The total DNA derived from signals does not directly reflect the structure and composition of microorganisms,and often leads to overestimate the viable microbial population and diversity,thus an accurate and feasible method is urgently required to be developed for screening viable microbes from complex environmental matrices.Propidium monoazide(PMA)is a light activation of DNA bound dye,which selectively permeates only into dead cells with compromised membrane integrity.Upon intercalation in the extracellular DNA and DNA from dead cells(collectively referred as relic DNA),relic DNA is irreversibly modified and inhibited for its PCR amplification.Sample pretreatment with PMA dye combined with quantitative PCR(qPCR)and high-throughput sequencing provides a powerful tool for fast and effectively screening environmental viable microbes.This paper reviewed the recent developments in understanding the detection of environmental viable microbes in sediments,soils and waters based on PMA dye,discussed the current problems,and proposed the strategies of PMA dye application;which would stimulate new ideas for future study of viable microbial communities and functional structures in complex environmental samples.