![]() ![]() 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. Gene amplicons (left to right): a, 16S rRNA b, ATPase c, sodA d, toxin e, atpD f, recA.Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. ST is based on the eight-gene MLST scheme of McDowell et al. acn31 (ST2, type IA 2) lane 7, strain 6609 (ST5, type IB) lane 8, strain VA3/4 (ST78, type IB) lane 9, strain 74874 (ST43, type IB) lane 10, strain PRP-38 (ST70, type IC) lane 11, strain PV66 (ST85, type IC) lane 12, strain 5/1/3 (ST107, type IC) lane 13, strain ATCC 11828 (ST27, type II) lane 14, strain VA2/9N (ST28, type II) lane 15, strain 6187 (ST30, type II) lane 16, strain 12S (ST32, type III) lane 17, strain Asn12 (ST33, type III) lane 18, strain Asn10 (ST81, type III) lane 19, Propionibacterium avidum strain 44067. Lane 1, strain hdn-1 (ST1, type IA 1) lane 2, strain PRP-60 (ST20, type IA 1) lane 3, strain 76793 (ST101, type IA 1) lane 4, strain Pacn33 (ST2, type IA 2) lane 5, strain P.acn17 (ST2, type IA 2) lane 6, strain P. acnes strains (except lane 19) representing different phylogroups and STs. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses.Ĭopyright © 2015, American Society for Microbiology. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. No cross-reactivity with isolates from other bacterial species was observed. acnes isolates previously characterized by MLST and representing types IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45), and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. ![]()
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