Quantitative stable isotope probing (qSIP) is a powerful tool, which links microbial taxon with functional metabolism in ecosystems and quantitatively determines the metabolic activity or growth rate of individual microbial taxa exposed to isotope tracers in the environment. qSIP technique employs quantitative PCR, high-throughput sequencing and stable isotope probing (SIP) techniques. The procedure involves adding labeled substrates to environmental samples for cultivation, separating labeled heavy fraction from unlabeled light fraction via isopycnic ultracentrifugation, making absolute quantification and sequencing analysis for microbial populations in all fractions, and then quantifying the isotope abundance of DNA involved in uptake and transformation based on the DNA density curve of unlabeled treatment and GC content. Here, we reviewed the rationale, data analysis and application of qSIP in microbial ecology, and discussed the existing problems and prospects of qSIP.
定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率。qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高速等密度梯度离心将被同位素标记的重链核酸与未被标记的轻链核酸进行分离,并对所有组分微生物类群进行绝对定量和测序分析,基于GC含量和未标记处理DNA密度曲线量化参与吸收转化的DNA同位素丰度。本文重点阐述qSIP的技术原理、数据分析流程及其在微生物生态学研究中的应用进展,并对该技术存在的问题进行了分析和展望。.
Keywords: high-throughput sequencing; isotope labelling; microbial function; microbial growth rate; microbial taxon.