Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR.
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.
Huijsdens
Xander W
XW
Department of Medical Microbiology, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.
Linskens
Ronald K
RK
Mak
Mariëtte
M
Meuwissen
Stephan G M
SG
Vandenbroucke-Grauls
Christina M J E
CM
Savelkoul
Paul H M
PH
D001422:Bacterial Adhesion; D001439:Bacteroides / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D003106:Colon / Q000382:microbiology*; D015169:Colony Count, Microbial; D004275:DNA, Ribosomal / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase