PDF(Pubmed)
Abstract：
The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson\'s r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.
摘要：
人类肠道微生物群是指共生存在于人类肠道系统中的多种微生物群落。改变的微生物群落与许多人类病理有关。然而,在实践中缺乏快速有效的方法来评估肠道微生物群特征.为了解决这个问题，我们建立了一个包含45个定量实时聚合酶链反应（qPCR）检测方法的评估系统，这些方法针对人群中患病率和/或丰度较高的肠道核心微生物。通过比较基因组分析，我们为45种核心微生物中的31种选择了新的物种特异性遗传标记和引物,这些核心微生物没有先前报道的特异性引物或其引物的特异性需要改进.我们全面评估了qPCR检测的性能，并证明它们显示出良好的灵敏度，选择性,和每个目标的定量线性。这些靶标的基因组DNA的检测限范围为0.1至1.0pg/µL。我们还证明了qPCR方法和宏基因组学下一代测序（mNGS）方法在分析22个人类粪便样品中选定细菌的丰度方面的高度一致性（Pearson'sr=0.8688，P<0.0001）。此外，我们使用qPCR定量了14个个体中这些核心微生物的动态变化（超过8周），大多数参与者都表现出相当大的稳定性，尽管存在显著的个体差异。总的来说,这项研究能够简单快速地定量人体肠道中的45种核心微生物，提供了一个有前途的工具来了解肠道核心微生物群在人类健康和疾病中的作用。关键点:•开发了一组原始qPCR测定以量化人类肠道核心微生物。•使用真实粪便样品评价qPCR测定并与mNGS比较。•该方法用于动态地描绘个体中的肠道核心微生物群。
