Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora

Ott, Stephan; Musfeldt, Meike; Ullmann, U.; Hampe, Jochen; Schreiber, Stefan

doi:10.1128/jcm.42.6.2566-2572.2004

Cited by 127 publications

(103 citation statements)

References 40 publications

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“…Different internal controls have been reported for qPCR-based quantification of bacterial abundances, including the Bacteroides genus (20), absolute DNA quantities (8), and 16S rDNAs (21)(22)(23). Considering that the proportion of Bacteroides varies among subjects of different enterotypes (24), and that the absolute DNA contained both bacterial DNAs and host DNAs, 16S rDNA could serve as a suitable internal control with conserved sequences uniformly distributed in most species.…”

Section: Discussionmentioning

confidence: 99%

Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

Liang

Chiu

Chen

et al. 2017

Clinical Cancer Research

260

251

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Purpose: Gut microbiota have been implicated in the development of colorectal cancer. We evaluated the utility of fecal bacterial marker candidates identified by our metagenome sequencing analysis for colorectal cancer diagnosis.Experimental Design: Subjects (total 439; 203 colorectal cancer and 236 healthy subjects) from two independent Asian cohorts were included. Probe-based duplex quantitative PCR (qPCR) assays were established for the quantification of bacterial marker candidates.Results: Candidates identified by metagenome sequencing, including Fusobacterium nucleatum (Fn), Bacteroides clarus (Bc), Roseburia intestinalis (Ri), Clostridium hathewayi (Ch), and one undefined species (labeled as m7), were examined in fecal samples of 203 colorectal cancer patients and 236 healthy controls by duplex-qPCR. Strong positive correlations were demonstrated between the quantification of each candidate by our qPCR assays and metagenomics approach (r ¼ 0.801-0.934, all P < 0.0001). Fn was significantly more abundant in colorectal cancer than controls (P < 0.0001), with AUROC of 0.868 (P < 0.0001). At the best cut-off value maximizing sum of sensitivity and specificity, Fn discriminated colorectal cancer from controls with a sensitivity of 77.7%, and specificity of 79.5% in cohort I. A simple linear combination of four bacteria (Fn þ Ch þ m7-Bc) showed an improved diagnostic ability compared with Fn alone (AUROC ¼ 0.886, P < 0.0001) in cohort I. These findings were further confirmed in an independent cohort II. In particular, improved diagnostic performances of Fn alone (sensitivity 92.8%, specificity 79.8%) and four bacteria (sensitivity 92.8%, specificity 81.5%) were achieved in combination with fecal immunochemical testing for the detection of colorectal cancer.Conclusions: Stool-based colorectal cancer-associated bacteria can serve as novel noninvasive diagnostic biomarkers for colorectal cancer.

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Section: Discussionmentioning

confidence: 99%

Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

Liang

Chiu

Chen

et al. 2017

Clinical Cancer Research

260

251

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“…Lanes represent Level reactions in the same order (1)(2)(3)(4)(5)(6)(7)(8)(9). Interpreted results are shown in the table Good sequencing results but poor BLAST alignment result, 2 indicates an independent repeat of sample processing on a different day e Different colony morphologies f H. pylori specific primers were used to confirm isolate, according to reference [8], while PCR product was generated with QUGP-F6.R3, no sequencing was attempted for this bacterium PA-SS-F and PA-SS-R [18] were utilized with this isolate.…”

Section: Dna Sequence Alignment and Bacterial Identificationmentioning

confidence: 99%

“…A robust method, required for bacterial identification, has been perused by several investigators [4,14,26,[36][37][38][39][40]; studies on universal and multiplex primers]. With the current number of eubacterial species surpassing 7166 species [41], the UM described here [42] should fulfil this requirement.…”

Section: Introductionmentioning

confidence: 99%

“…To solve this problem, investigators [4,17,20,25,27,[29][30][31] sought universal primers. Unfortunately, ''Universal Primers'' do not live up to their name since they do not cover all bacteria [30].…”

Section: Introductionmentioning

confidence: 99%

“…Historically, identification and classification of eubacteria are based on phenotypic characteristics [1][2][3][4]. Early molecular techniques used in bacterial classification were based on GC content [5], plasmid profiling, and compatibility to genetic transformation [6].…”

Section: Introductionmentioning

confidence: 99%

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A Universal Method for the Identification of Bacteria Based on General PCR Primers

Barghouthi

2011

Indian J Microbiol

103

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The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.

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Molecular and “Omics” Techniques for Studying Gut Microbiota Relevant to Food Animal Production

Gong

Yang

Khafipour

2018

Molecular Techniques in Food Biology

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Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora

Cited by 127 publications

References 40 publications

Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Molecular and “Omics” Techniques for Studying Gut Microbiota Relevant to Food Animal Production

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