In this study an approach that combines the specificity of fluorescent oligonucleotide probes with the sensitivity of PCR was used. E. coli and B. vulgatus were used for evaluation of the 5′ nuclease PCR assay as a tool to identify and quantify intestinal bacteria. Both bacteria are prominent normal gut bacteria that play an important role in the maintenance of a healthy gut microflora.
Conventional PCR has several disadvantages. These include the sensitivity of the assay to inhibition by substances present in the sample to be analyzed, the limitation of a small sample input, and the possibility of nonspecific binding of the primers or the probe. Finally, the PCR assay is very susceptible to contamination. The 5′ nuclease PCR assay (real-time PCR) solves several of these problems. In real-time PCR two primers and one probe are used, and a fluorescent signal can be generated only when all three are bound to the DNA at the correct primer and probe locations, which greatly reduces the risk of nonspecific binding. The assay is performed in a closed-tube system, and no post-PCR handling is necessary. This reduces the risk of contamination.
Specificity, a key factor for accurate quantification of the bacteria of interest, was determined by performing the reaction with a sample consisting of a mixture of genomic DNA isolated from eight different bacterial species commonly found in the intestinal microflora. In addition, the specificity was also tested by using DNA purified from closely related species: Salmonella serovar Typhimurium for E. coli and B. fragilis for B. vulgatus. Nonspecific signals were not detected in these reactions. The specificity of our approach is hampered only by the availability of species-specific DNA sequences. In this respect, 16S rDNA sequences are not always satisfactory. This is illustrated by the homology between the 16S rDNA sequences of E. coli, E. vulneris, and Shigella. This homology is so close that we were not able to design a primer-probe combination that would distinguish these bacteria. More and more sequence information is continuously becoming available, however, and sequences which are truly specific for each bacterial species will be available in the near future. Such specific sequences can be used, like we used the 16S rDNA sequences, to design primers and probes in cases in which 16S rDNA-based discrimination is not possible.
In the first set of experiments, a positive signal was also obtained with samples that in principle did not contain E. coli
DNA. We were able to show that this was due to contamination, probably caused by traces of E. coli
DNA present in the Taq
polymerase and/or the UNG enzyme. DNase I treatment of the universal PCR master mixture eliminated this signal, which confirmed this hypothesis. Corless et al. (3
) previously mentioned the E. coli
contamination of Taq
The assay proved very sensitive: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus bacteria could be detected. The difference in sensitivity cannot be explained by differences in the numbers of copies of the 16S rRNA genes, since both species have seven copies. It is possible that the DNA extraction efficiency or anaerobic culture conditions may have influenced the sensitivities of the assays for E. coli and B. vulgatus and resulted in differences in sensitivities.
Compared to quantification by determination of viable counts, the quantification of bacteria by real-time PCR yielded approximately 100-fold higher numbers of E. coli. This appears plausible, since all DNA is amplified by PCR, including DNA from dead bacteria. The median percent cultivatable bacteria was 1%, indicating that a high percentage of the bacteria that adhere to the intestinal mucosa are dead or viable but not cultivatable.
We used the real-time PCR to investigate bacteria that adhere to the gastrointestinal mucosa, not the bacteria present in fecal samples (6
). We did this because in future studies we aim to evaluate the role of this adherent flora in the inflammation of the mucosa that is characteristic of inflammatory bowel diseases.
A problem with PCR-based assays is that the reaction is very susceptible to inhibitory factors that can be present in the samples under study. To determine whether inhibition was a problem, we spiked biopsy samples with as little as 5 CFU of E. coli. These low numbers were accurately detected. This indicated that no inhibition factors seem to be influencing the quantitative PCR results for mucosal biopsy specimens or that inhibitory factors were removed with the DNeasy tissue kit (Qiagen) used for DNA isolation.
We found that the reproducibility of the quantitative experiments was over 99%, based on multiple replicate PCR runs. The 5′ nuclease PCR assay is therefore an accurate method that can be used to gain a better insight into the actual in vivo composition of the microflora that adheres to the intestinal mucosa.
Investigation of this microflora is crucial for obtaining an understanding of the role of the microflora in gut health but also an understanding of the role of the microflora in inflammatory bowel diseases like Crohn's disease and ulcerative colitis.
Many factors, such as differences in nutrition or use of antibiotics, can have an effect on the gastrointestinal microflora. The 5′ nuclease PCR assay in combination with a high-throughput screening system makes the study of these effects on the microflora of each individual possible.