Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes

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.

FIG. 1.

Beginning and ending points of RDP bacterial 16S rRNA sequences in E. coli coordinates. The graphs show the number of sequences beginning or ending at a given point and the total number of sequences that include (cover) the position. (A) Region corresponding to E. coli positions 1 to 36, which includes the 27f primer-binding site; (B) region corresponding to E. coli positions 1484 to 1519, which includes the 1492r primer-binding site.

FIG. 2.

Change in the abundances of Lactobacillus and Gardnerella rRNA genes during linear amplification with three different 27f primer formulations. Amplification was carried out with a human vaginal DNA sample at the following three different annealing temperatures: 48°C, 54°C, and 60°C. Each sample was analyzed by qPCR using primers specific for Lactobacillus (left panels) and Gardnerella (right panels). Data are relative fluorescence units of DNA in the 26th cycle of qPCR. The fluorescence units are normalized to “two strands” at zero cycles of linear amplification. Each data point represents the average of results from three independent experiments.

FIG. 3.

Change in Lactobacillus and Gardnerella sequence ratios after PCR amplification using each of three 27f primer formulations and the 1492r primer. The values plotted are ΔΔC_Ts, the change in the Lactobacillus C_T minus the Gardnerella C_T resulting from 23 cycles of PCR amplification of the DNA samples. Values are the means and the corresponding standard errors from three experiments based on the same data as are shown in Table 2.