Environ Microbiol. 2014 Aug;16(8):2389-407. doi: 10.1111/1462-2920.12350.
Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.
定量聚合酶链式反应（qPCR）和群落指纹分析方法，如末端限制性片段长度多态性（T-RFLP）分析，是研究微生物群落结构的理想技术。与基于域的引物相比，基于门和纲特异性的引物可以提供更高的灵敏度和系统发育分辨率。迄今为止，已经有一些针对 16S 核糖体 RNA 基因的基于门和纲特异性的引物被发表。然而，许多这些引物在 PCR 中对非靶标细菌的区分能力较低。在本研究中，我们通过计算机模拟和特定 PCR 对某些已发表的引物的精确性进行了评估。我们通过结合公共数据集（SILVA SSU Ref 102 NR）中的序列信息和手动引物设计，设计了新的 qPCR 和 T-RFLP 引物对（用于α变形菌纲和β变形菌纲以及拟杆菌门、厚壁菌门和放线菌门）。我们通过使用上述组的分离株和环境土壤样本和人粪便样本的克隆文库进行 PCR 评估了这些引物对。通过理论和实际评估观察到，本研究中开发的引物比以前发表的引物具有更高的精度，从而可以更深入地了解微生物群落动态。
