Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

Kommedal, Øyvind; Simmon, Keith E.; Karaca, Dilek; Langeland, Nina; Wiker, Harald G.

doi:10.1128/jcm.06269-11

Cited by 54 publications

(36 citation statements)

References 21 publications

(25 reference statements)

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“…This factor may be responsible for the cross-reactivity of 16S primers with human DNA. Kommendal et al (24) described mixed chromatograms or amplification of fragments corresponding to part of human chromosome 9. Our primers can also lead to nonspecific amplification of 150 bp of human chromosome 1, so these cases were classified as negative (data not shown).…”

Section: Discussionmentioning

confidence: 99%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI.

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Section: Discussionmentioning

confidence: 99%

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Plouzeau

Bémer²,

Valentin

et al. 2015

J Clin Microbiol

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“…All samples were screened for bacterial DNA using a real-time universal 16S rRNA-PCR, performed as described previously (6). A sample was defined as positive if the fluorescence threshold cycle (C T ) was reached three or more cycles ahead of the negative control (⌬C T Ͼ 3) (5, 7).…”

Section: Methodsmentioning

confidence: 99%

Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

et al. 2014

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m Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture-and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop. O ur understanding of polymicrobial infections has been hindered by our limited possibilities for describing them. Recent investigations of bacterial brain abscesses using universal amplification of the bacterial 16S rRNA gene, followed by Sanger sequencing of cloned amplicons, have revealed that only a fraction of the bacteria present are identified by culture (1, 2). Nevertheless, this approach has limitations when it comes to detecting smaller subpopulations in a multispecies community, unless very high numbers of clones are sequenced (3). This is problematic, since the species structure of an abscess may change over time and pathogens important for establishing the infection potentially remain at only low concentrations in the more mature abscesses. Furthermore, the species that are important for maintaining and expanding the abscess might primarily exist close to the abscess wall and do not necessarily dominate in the pus obtained by aspiration. Rapid development within the field of massive parallel sequencing technologies (MPS) is about to provide the diagnostic laboratories with tools that can characterize even the most complex microbial communities. The aim of the present study was to use recent adva...

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“…Total bacterial DNA was quantified by quantitative PCR using the following 16S rRNA universal primers: 16SDPO-forward 5 0 -AGAGTTTGATCMTGGCTCA-I-I-I-I-I-AACGCT-3 0 ; 16SDPO-reverse 5 0 -CGCGGCTGCTGGCA-I-I-I-A-I-TTRGC-3 0 or EUBAC-forward, 5 0 -TCCTACGGGAGGCAGCAGT-3 0 ; EUBAC-reverse, 5 0 -GGACTACCAGGGTATCTAATCCTGTT -3 0 as previously reported. 76,77 Quantitative PCR for total bacteria were carried out using a SYBR Premix EX Taq TM and ABI PRISM 7300 quantitative PCR System (Applied Biosystems, Waltham, MA, USA).…”

Section: Dna Extraction and Quantification Of Bacteria By Quantitativmentioning

confidence: 99%

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

et al. 2016

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(2016) Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis, Autophagy, 12:8, 1390-1403, DOI: 10.1080/15548627.2016 ABSTRACT Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mfs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 b release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD C :NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mfs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mfs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mfs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mfs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.

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Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

Cited by 54 publications

References 21 publications

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

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