Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes

TLDR

rRNA‑based studies rely on faithful amplification of microbial community genes from DNA samples. The study analyzes and reevaluates common primers for amplifying bacterial 16S rRNA genes and proposes a new forward primer formulation. The authors compared the new primer to two common alternatives using linear amplification and PCR with the 1492 reverse primer under community clone‑library conditions. The new primer better preserved Lactobacillus and Gardnerella rRNA gene ratios in vaginal samples, especially under stringent conditions, while maintaining amplification efficiency and specificity.

Abstract

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.