Inglis G Douglas, Thomas Matthew C, Thomas Dallas K, Kalmokoff Martin L, Brooks Stephen P J, Selinger L Brent
Agriculture and Agri-Food Canada, 5403-1st Ave S, Lethbridge, AB T1J 4B1, Canada.
J AOAC Int. 2012 Jan-Feb;95(1):5-23. doi: 10.5740/jaoacint.sge_inglis.
The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.
肠道是一个极其丰富的生态系统，包含微生物之间复杂的相互作用，受宿主因素、摄入的食物和液体影响。表征肠道微生物群是当前一个活跃的研究领域。有多种基于分子的方法可用于表征肠道微生物群，但所有方法都有相对的优点以及显著的缺点。研究人员认识到这些方法的局限性，并采取适当措施减轻其弱点很重要。在此，我们讨论用于监测肠道细菌的方法，包括：（i）传统克隆文库；（ii）使用下一代平行测序技术的直接测序；（iii）变性梯度凝胶电泳和温度梯度凝胶电泳；（iv）末端限制性片段长度多态性分析；（v）荧光原位杂交；以及（vi）定量PCR。此外，我们还讨论实验设计、样本采集与储存、DNA提取、基因靶点、PCR偏差以及减少PCR偏差的方法。
