![]() ![]() fragilis toxin ( bft) gene from cultured ETBF DNA, and from matched luminal and faecal stool samples. The aim of this study was to compare the different methods previously described (standard PCR, SYBR green and TaqMan quantitative PCR), in addition to digital PCR, in detecting the B. Digital PCR (dPCR) is a third generation PCR platform that allows direct absolute quantification without the need for standard curve generation 15, and it has also been shown to tolerate PCR inhibitors better than qPCR 16. Absolute quantitation using qPCR requires the use of a standard dilution series, and the accuracy of qPCR decreases with lower target abundance, due to the logarithmic nature of PCR. The use of standard (end-point) PCR has been described in several studies to detect ETBF in faecal samples, and a recent study by Sears’ group has used touch-down PCR and quantitative PCR (qPCR) to detect faecal ETBF with greater sensitivity 14. Detection of low abundance bacterial species may also be hindered by the dilutive effect of faecal matter and other more abundant bacteria, and the inhibitory effects of bile salts, and faecal proteins and enzymes on the detection methods used 13. A recent study by Li et al., has demonstrated a higher level of microbial diversity in luminal stool and tissue samples from the duodenum, compared to samples taken from the rectum, and to faecal samples 12. The majority of reports published to date regarding the gut microbiome have looked exclusively at faecal stool samples. However, bacterial detection in faecal stool samples may not be a true reflection of the microbial population at different locations in the gut. ![]() The presence of ETBF has been reported in the stool samples of colorectal cancer patients in several studies 1, 3, 11, and a sensitive method of faecal detection of these bacteria may be important in future colorectal screening programmes. Although the exact mechanisms by which ETBF contributes to the development of colorectal carcinogenesis have yet to be elucidated, the presence of these bacteria may represent an early indicator/risk factor of colorectal neoplasia. ETBF has also been shown to promote chronic inflammation in the gut by stimulating secretion of the pro-inflammatory cytokine, IL-8 9, and by Stat3 activation 10, which may also drive tumorigenesis. In vitro studies have shown that ETBF results in the cleavage of E-cadherin 5, 6, resulting in β-catenin signalling and colonic epithelial cell proliferation 7 that promotes epithelial-mesenchymal transition, a process involved in carcinogenesis. fragilis toxin (BFT) 1, 2 or 3, has been reported in up to 30% of the population 4. Colonization with ETBF that carry a gene coding for a metalloprotease, B. fragilis is a gram negative commensal bacteria and colonic carriage has been reported in up to 80% of people. Prominent among these bacterial species is enterotoxigenic Bacteroides fragilis (ETBF). The association between gut colonization with particular bacterial species, and the development of colorectal cancer, has been reported by our group and several others 1, 2, 3. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples. For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively ( p < 0.001). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). fragilis toxin ( bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. However, differences in carriage rates are seen with various testing methods and sampling sites. Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. ![]()
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