Molecular ecology of marine sediments: determination of Real-Time PCR efficiency for quantifying microbial cells

Abstract

Polymerase chain reaction (PCR) efficiency is the rate at which a PCR amplicon is generated. More efficient amplification of the DNA fragment (amplicon) will generate more products in fewer cycles, thus improving accuracy and sensitivity of quantitative PCR. The efficiency of Real-Time PCR to detect and quantify microbial cells was compared in marine sediment samples of different origin and chemical composition, and in standard samples. Real-Time PCR efficiencies of marine sediment samples ranged from 1.48±0.1 to 1.83±0.1 and were significantly different from those of standard samples, most probably due to different concentrations of PCR inhibitors. The Real-Time PCR efficiency was higher using species-specific primers (>1.7), and lower using universal primers (<1.7). Generally, when the PCR efficiency was higher, its detection limit was lower. In addition, the sensitivity of the Real-Time capillary assay over the traditional assay was generally greater. We suggest that for quantifying microbial cells in marine sediment samples using Real-Time PCR, standard curves should be constructed for both the standard and sediment samples, and a correction factor should be applied. For qualitative PCR a real-time capillary assay should be used for the detection of small quantities of DNA.