Department of Biology and Biochemistry, University of Houston, Houston, TX, United States.
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United States.
Front Cell Infect Microbiol. 2018 Jul 12;8:237. doi: 10.3389/fcimb.2018.00237. eCollection 2018.
Changes in the population levels of specific bacterial species within the gut microbiome have been linked to a variety of illnesses. Most assays that determine the relative abundance of specific taxa are based on amplification and sequencing of stable phylogenetic gene regions. Such lab-based analysis requires pre-analytical sample preservation and storage that have been shown to introduce biases in the characterization of microbial profiles. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification method that employs commercially available, easy-to-use freeze-dried enzyme pellets that can be used to analyze specimens rapidly in the field or clinic, using a portable fluorometer. Immediate analysis of diverse bacterial communities can lead to a more accurate quantification of relative bacterial abundance. In this study, we discovered that universal bacterial 16S ribosomal DNA primers give false-positive signals in RPA analysis because manufacturing host DNA is present in the RPA reagents. The manufacturer of RPA reagents advises against developing an RPA assay that detects the presence of due to the presence of contaminating DNA in the reaction buffer (www.twistdx.co.uk/). We, therefore, explored four strategies to deplete or fragment extraneous DNA in RPA reagents while preserving enzyme activity: metal-chelate affinity chromatography, sonication, DNA cleavage using methylation-dependent restriction endonucleases, and DNA depletion using anti-DNA antibodies. Removing DNA with anti-DNA antibodies enabled the development of a quantitative RPA microbiome assay capable of determining the relative abundance of the physiologically-important bacterium in human feces.
肠道微生物组中特定细菌物种的种群水平变化与多种疾病有关。大多数确定特定分类相对丰度的检测方法都是基于稳定的系统发育基因区域的扩增和测序。这种基于实验室的分析需要在分析前保存和储存样本，已证明这会导致对微生物特征的描述产生偏差。重组酶聚合酶扩增（RPA）是一种等温核酸扩增方法，它使用市售的、易于使用的冻干酶粉，可以使用便携式荧光计在现场或诊所中快速分析标本。对不同细菌群落的即时分析可以更准确地定量相对细菌丰度。在这项研究中，我们发现通用细菌 16S 核糖体 DNA 引物在 RPA 分析中会产生假阳性信号，因为 RPA 试剂中存在制造宿主 DNA。RPA 试剂制造商建议不要开发检测 的 RPA 检测方法，因为反应缓冲液中存在污染的 DNA（www.twistdx.co.uk/）。因此，我们探索了四种策略来去除或断裂 RPA 试剂中的外源 DNA，同时保持酶活性：金属螯合亲和层析、超声处理、使用甲基化依赖性限制内切酶切割 DNA 以及使用抗 DNA 抗体进行 DNA 耗竭。使用抗 DNA 抗体去除 DNA 使开发一种定量 RPA 微生物组检测方法成为可能，该方法能够确定生理相关细菌 在人类粪便中的相对丰度。
