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Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.

ABSTRACT: Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LAMP assays to detect these bacteria and evaluated the specificity and sensitivity of these assays. The specificities of the primers for these bacteria were examined using various oral bacteria and various reaction times. The lower detection limits of the 60-min LAMP reaction without loop primers were 1 microg/tube for P. gingivalis, 10 fg/tube for T. forsythia, and 1 ng/tube for T. denticola. Addition of the loop primers for each bacterium improved the detection specificities and sensitivities by several magnitudes. Furthermore, LAMP assays were applied to the rapid detection of these periodontal pathogens in clinical specimens, and the results were compared with those of conventional PCR detection. The results of the LAMP assays corresponded to those of conventional PCR assays. These results indicate that the LAMP assay is an extremely rapid, highly sensitive, specific method. This method is very useful for the rapid detection of periodontopathic bacteria and the diagnosis of periodontal disease.

SUBMITTER: Yoshida A

PROVIDER: S-EPMC1153746 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.

Yoshida Akihiro A Nagashima Shiori S Ansai Toshihiro T Tachibana Masayo M Kato Hiroaki H Watari Hajime H Notomi Tsugunori T Takehara Tadamichi T

Journal of clinical microbiology 20050501 5

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the rapid detection of the major periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. The LAMP method amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. In this study, we initially designed the primers for LA ...[more]

PMID: 15872275

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Project description:Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species, such as Porphyromonas gingivalis and Treponema denticola contribute to disease progression. P. gingivalis and T. denticola are co-localized in subgingival plaque and have been previously shown to exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola, although not considered as a classic virulence factor, may be involved in synergistic biofilm development between P. gingivalis and T. denticola. We determined the role of T. denticola motility in polymicrobial biofilm development using an optimized transformation protocol to produce two T. denticola mutants targeting the motility machinery. These deletion mutants were non-motile and lacked the gene encoding the flagellar hook protein of the periplasmic flagella (ΔflgE) or a component of the stator motor that drives the flagella (ΔmotB). The specificity of these gene deletions was determined by whole genome sequencing. Quantitative proteomic analyses of mutant strains revealed that the specific inactivation of the motility-associated gene, motB, had effects beyond motility. There were 64 and 326 proteins that changed in abundance in the ΔflgE and ΔmotB mutants, respectively. In the ΔflgE mutant, motility-associated proteins showed the most significant change in abundance confirming the phenotype change for the mutant was related to motility. However, the inactivation of motB as well as stopping motility also upregulated cellular stress responses in the mutant indicating pleiotropic effects of the mutation. T. denticola wild-type and P. gingivalis displayed synergistic biofilm development with a 2-fold higher biomass of the dual-species biofilms than the sum of the monospecies biofilms. Inactivation of T. denticola flgE and motB reduced this synergy. A 5-fold reduction in dual-species biofilm biomass was found with the motility-specific ΔflgE mutant suggesting that T. denticola periplasmic flagella are essential in synergistic biofilm formation with P. gingivalis.

Project description:BackgroundTannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the "red complex" of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network.ResultsWe investigated T. forsythia's peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type.ConclusionT. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

Dataset Information

Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.

Publications

Loop-mediated isothermal amplification method for rapid detection of the periodontopathic bacteria Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola.

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