在人类便样本中的细菌菌株的高度准确和敏感的绝对量化
Fuyong Li1,2, Junhong Liu3, María X Maldonado-Gómez4
1Department of Animal Science and Technology, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, China. fuyong@zju.edu.cn.
Microbiome
|September 7, 2024
概括
定量PCR (qPCR) 与滴滴数字PCR (ddPCR) 相比,为精确测量便样本中的细菌菌株提供了一种优越的方法. 这项研究提供了一个优化的qPCR协议,用于在菌株水平上准确量化肠道细菌.
科学领域:
- 微生物学 微生物学
- 分子生物学分子生物学
- 基因组学就是基因组学.
背景情况:
- 下一代测序 (NGS) 提供了应变级别的分辨率,但在量化,检测限制和数据组合性方面存在局限性.
- 精确量化肠道微生物群中的特定细菌菌株对于理解宿主-微生物相互作用至关重要.
研究的目的:
- 系统地比较定量PCR (qPCR) 和滴滴数字PCR (ddPCR) 以在人类便样本中绝对量化Limosilactobacillus reuteri菌株.
- 开发和验证一种针对菌株特定的qPCR测定设计和在便样本中的实施的优化协议.
主要方法:
- 特定菌株的原料是为Limosilactobacillus reuteri而设计的.
- 使用定量PCR (qPCR) 和滴滴数字PCR (ddPCR) 进行绝对量化.
- 使用了基于套件的DNA隔离方法.
- 开发和验证了QPCR测试设计和校准的逐步协议.
主要成果:
- 在使用基于套件的DNA隔离时,qPCR表现出与ddPCR相似的灵敏度和可重复性,检测极限 (LOD) 约为10^4细胞/g便.
- qPCR表现出更广泛的动态范围,比ddPCR更快,更具成本效益.
- 优化后的qPCR协议在增样本中达到大约10^3细胞/g便的检测极限,在人体便样本中准确量化L. reuteri菌株,优于NGS方法.
结论:
- 定量PCR (qPCR) 与基于套件的DNA提取相结合是准确,在便样本中精确量化肠道细菌菌株水平的首选方法.
- 开发的协议可以为各种细菌种群和样本类型设计高度敏感的,菌株特定的qPCR测定.
- 这种方法在细菌菌株量化方面的灵敏度,动态范围和成本效益方面比NGS具有显著的优势.
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