Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes
Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes
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DOI：
10.1128/aem.02272-07
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发表时间：
2008-04-01
影响因子：
4.4
通讯作者：
Olsen, Gary J.
中科院分区：
文献类型：
--
作者：
Frank, Jeremy A.;Reich, Claudia I.;Olsen, Gary J.
基于rrna的研究已经成为评估微生物群落最常用的方法，它依赖于对原始DNA样本中相应基因的忠实扩增。我们在这里报告了一项分析和重新评估常用的引物，用于扩增细菌16S rRNA基因（根据大肠杆菌rRNA编号）第27和1492位之间的DNA。我们提出了一个正向引物（27f）的配方，其中包括通常不存在的三个序列。我们将我们提出的配方与两种常见的替代方案进行比较，方法是使用线性扩增（提供独立于反向引物的评估），并在适合制作社区rRNA基因克隆文库的PCR条件下与1492反向引物（1492r）结合使用。对于人类阴道样本的DNA分析，我们的配方能够更好地保持乳酸菌与加德纳菌的原始rRNA基因比例，特别是在严格的扩增条件下。由于我们的27f配方仍然相对简单，有7个不同的引物序列，因此总体扩增效率和特异性的损失最小。
rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification-providing an assessment that is independent of a reverse primer-and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.