Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR

Abstract

The use of real-time quantitative PCR (5' nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5' nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5' nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.

FIG. 1.

Amplification sensitivity of 5′ nuclease PCR assay for E. coli. DNA isolated from log-phase bacteria was used in serial 10-fold dilutions from 135,000 bacteria to 1 bacterium per reaction mixture. NTC, no-template control; ΔRn, fluorescence intensity after subtraction of background signal.

FIG. 2.

Quantification of B. vulgatus adherent to mucosal biopsy specimens from healthy persons. DNA isolated from biopsy specimens was used in a 5′ nuclease PCR assay with the primer-probe set for B. vulgatus. A range of known concentrations of bacteria was amplified and plotted (black crosses) as a standard curve as a reference for the quantification of B. vulgatus in mucosal biopsy specimens. For quantification, a standard curve was obtained by plotting the points of the fluorescent signals that cross the threshold line (C_t values). Each dot represents the result of an amplification of DNA from a mucosal biopsy specimen. A mean equivalent of 4.2 × 10⁶ B. vulgatus CFU was found in mucosal biopsy specimens. R was equal to 0.999, and the slope was −3.412.