Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

TLDR

Accurate bacterial load estimation depends on using a DNA standard that reflects the predominant bacteria in a habitat, because 16S rDNA copy number varies with species and growth phase. The study designs and evaluates universal 16S rDNA primers and probe for estimating total bacterial load by real‑time PCR. The method was validated by quantifying bacteria in artificial mixtures and clinical carious dentine samples using the universal primer/probe set. The universal system showed broad specificity across 34 species and, when applied to carious dentine, estimated anaerobic bacterial loads 40‑fold higher than culture, highlighting real‑time PCR’s utility.

Abstract

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.