Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples.

IF 13.8 1区 生物学 Q1 MICROBIOLOGY Microbiome Pub Date : 2024-09-07 DOI:10.1186/s40168-024-01881-2
Fuyong Li, Junhong Liu, María X Maldonado-Gómez, Steven A Frese, Michael G Gänzle, Jens Walter
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Abstract

Background: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.

Results: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).

Conclusions: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. Video Abstract.

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高精度、高灵敏度地绝对量化人类粪便样本中的细菌菌株。
背景:下一代测序(NGS)方法彻底改变了肠道微生物组研究,可提供菌株级分辨率,但这些技术存在局限性,即只能半定量,检测限高,生成的数据是组成性的。本研究旨在系统比较定量 PCR(qPCR)和液滴数字 PCR(ddPCR)对人类粪便样本中芦特氏乳杆菌菌株的绝对定量,并为粪便样本中细菌菌株的绝对定量制定优化方案:使用针对 L. reuteri 17938 的菌株特异性 PCR 引物,ddPCR 的重现性稍好,但使用基于试剂盒的 DNA 分离方法时,qPCR 的重现性和灵敏度(检测限 [LOD] 约为 104 个细胞/克粪便)及线性度(R2 > 0.98)与之相当。基于这些发现,我们得出结论:在粪便样本中细菌菌株的绝对定量方面,qPCR 比 ddPCR 更有优势。我们为设计菌株特异性 qPCR 检测方法提供了一个优化且简单易学的分步方案,从基因组序列引物设计到 PCR 系统校准。通过对该方案的验证,我们设计出了针对两种 L. reuteri 菌株(PB-W1 和 DSM 20016 T)的 PCR 检测方法,从而实现了高度准确的 qPCR,在粪便样本中的检测限约为 103 个细胞/克粪便。在一项人体试验中,我们将菌株特异性 qPCR 检测方法应用于从接受活的 L. reuteri PB-W1 或 DSM 20016 T 的人体受试者采集的粪便样本,结果表明这两种菌株的定量准确度高,检测灵敏度高,与 NGS 方法(16S rRNA 基因测序和全元基因组测序)相比,LOD 更低,动态范围更广:根据我们的分析,我们认为使用试剂盒提取 DNA 的 qPCR 是对粪便样本中的肠道细菌进行菌株水平精确定量的最佳方法。所提供的分步方案将使科学家们能够设计出高灵敏度的菌株特异性 PCR 系统,不仅能准确量化 L. reuteri 细菌菌株,还能在广泛的应用和样品类型中准确量化其他细菌类群。视频摘要。
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来源期刊
Microbiome
Microbiome MICROBIOLOGY-
CiteScore
21.90
自引率
2.60%
发文量
198
审稿时长
4 weeks
期刊介绍: Microbiome is a journal that focuses on studies of microbiomes in humans, animals, plants, and the environment. It covers both natural and manipulated microbiomes, such as those in agriculture. The journal is interested in research that uses meta-omics approaches or novel bioinformatics tools and emphasizes the community/host interaction and structure-function relationship within the microbiome. Studies that go beyond descriptive omics surveys and include experimental or theoretical approaches will be considered for publication. The journal also encourages research that establishes cause and effect relationships and supports proposed microbiome functions. However, studies of individual microbial isolates/species without exploring their impact on the host or the complex microbiome structures and functions will not be considered for publication. Microbiome is indexed in BIOSIS, Current Contents, DOAJ, Embase, MEDLINE, PubMed, PubMed Central, and Science Citations Index Expanded.
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