The isolation chip method(iChip)provides a novel approach for culturing previously uncultivable microorganisms;this method is currently limited by the user being unable to ensure single-cell loading within individual ...The isolation chip method(iChip)provides a novel approach for culturing previously uncultivable microorganisms;this method is currently limited by the user being unable to ensure single-cell loading within individual wells.To address this limitation,we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip(FACS-iChip)to effectively mine microbial dark matter in soils.This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing.Results showed that the FACS-iChip achieved a culture recovery rate of almost 40%and a culture retrieval rate of 25%.Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates,only six genera could be identified via 16S rRNA gene amplification.This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology.We,therefore,conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial‘dark matter’mining.展开更多
基金This research was financially supported by the National Natural Science Foundation of China(41991334)the Zhejiang Provincial Natural Science Foundation of China(LD19D060001,LQ20C030006)the China Postdoctoral Science Foundation(2019M652097).
文摘The isolation chip method(iChip)provides a novel approach for culturing previously uncultivable microorganisms;this method is currently limited by the user being unable to ensure single-cell loading within individual wells.To address this limitation,we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip(FACS-iChip)to effectively mine microbial dark matter in soils.This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing.Results showed that the FACS-iChip achieved a culture recovery rate of almost 40%and a culture retrieval rate of 25%.Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates,only six genera could be identified via 16S rRNA gene amplification.This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology.We,therefore,conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial‘dark matter’mining.
