Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque

Fujimoto, C.; Maéda, Hiroshi; Kokeguchi, Susumu; Takashiba, Shogo; Nishimura, Fusanori; Arai, Hideo; Fukui, Kiichi; Murayama, Yoji

doi:10.1034/j.1600-0765.2003.02607.x

Cited by 54 publications

(48 citation statements)

References 26 publications

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“…The use of PCR and DGGE combined with GC-rich primers at the 5′ end has been shown to identify almost all of the sequence variations in a sample [20] .This method allows direct identification and relative abundance of the bacterial species present in a sample, without the risks of inability to directly culture by conventional methods only [18]. Some deficiencies of DGGE are that multiple species may be present in one band when examining complex microbial communities [21], but PCR amplification and identification were undertaken of the samples taken at each stage in the construction process to enable identification of the species present. These were found not to be salivary in origin, which corroborates the DGGE profiles.…”

Section: Discussionmentioning

confidence: 99%

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Microbial contamination of laboratory constructed removable orthodontic appliances

et al. 2014

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms AbstractObjectives: To determine whether laboratory constructed removable orthodontic appliances are free from microbial contamination prior to clinical use and to evaluate the dental hospital cross-infection procedures to ensure patient-derived contamination does not enter the construction process, thereby propagating a cycle of cross-contamination. Materials and Methods:The construction process of removable orthodontic appliances from three individuals was evaluated at every stage, from impression to final delivery of the appliance using molecular microbiological techniques. The bacterial profiles at each stage of appliance construction were obtained using Denaturing Gradient Gel Electrophoresis, along with the bacterial profiles of the three participants' saliva. This enabled the bacterial profiles found at each stage of construction to be compared directly with the saliva of the person for whom the appliance was being constructed. Bacteria were identified at each stage by using 16S rDNA PCR amplification and sequence phylogeny. Results:There was no evidence of bacterial cross-contamination from patients to the laboratory. The current process of disinfection of impression appears to be adequate. Contamination was found on the final removable appliances (0.97x10 2 -1.52x10 3 cfu ml -1) and this contamination occurred from within the laboratory itself. Conclusions:Every effort is made to reduce potential cross-infection to patients and dental professionals. Newly constructed removable appliances to be shown not to be free from contaminated with bacteria prior to clinical use, but this contamination is environmental. Further studies would be required to determine the level of risk this poses to patients.Clinical significance: Dental professionals have a duty of care to minimise or eradicate potential risks of crossinfection to patients and other members of the team. To date much less attention has been paid to contamination from the orthodontic laboratory so contamination and infection risks are unknown.

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Section: Discussionmentioning

confidence: 99%

“…Overestimation can also be a problem when using PCR-amplified 16S rDNA due to the possibility of heterogeneity of the 16S rDNA amplicons. This is reduced using specific touchdown protocols as employed in this study, which reduces spurious amplification during PCR [21].…”

Section: Discussionmentioning

confidence: 99%

Microbial contamination of laboratory constructed removable orthodontic appliances

et al. 2014

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“…Modern molecular techniques, particularly those based on amplifying 16S rRNA genes, include cloning and sequencing, quantitative DNA-DNA hybridization (checkerboard), denaturing gradient gel electrophoresis (DGGE), and real-time PCR. These methods have superseded conventional culture-based methods for quantifying the microbiota of healthy and diseased oral cavity (9,12,13,28). But to date, the implementation of these molecular techniques is still restricted to central laboratories because of the high equipment cost and the requirement for highly trained personnel.…”

Section: Methodsmentioning

confidence: 99%

<b>PCR-dipstick DNA chromatography for profiling of a subgroup of cariesassociated bacterial species in plaque from healthy coronal surfaces and periodontal </b><b>pockets </b>

et al. 2016

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The onset of plaque-mediated disease, including dental caries and periodontal diseases, is highly associated with compositional change of the resident microflora from the ecological perspective. As specific bacterial profiles have been linked to different disease stages, microbial compositional measurements might therefore have great value for clinical diagnosis. Previously we have reported a dry-reagent strip biosensor-PCR-dipstick DNA chromatography, which utilized molecular recognition of oligonucleotides and biotin-streptavidin, and the optical property of colored microspheres, for semiquantifying a five-membered subgroup of caries-associated bacterial species in supragingival plaque from healthy coronal surfaces of teeth. The present study aimed to evaluate this technique's ability to differentiate microflora by comparing the subset profiles. Sixteen subgingival plaque specimens were pooled from periodontal pockets and analyzed for the composition of Streptococcus mutans, Streptococcus sobrinus, Scardovia wiggsiae, Actinomyces sp. and Veillonella parvula. Detection frequencies, relative abundance of each bacterial species, and the five-membered bacterial profiles were compared between supra-and subgingival groups. The supragingival plaque harbored significantly more of the tested species and higher amount of Actinomyces sp. and V. parvula. In subgingival plaque, the predominance was obscured, since several highly overlapped profiles were found at comparable frequencies. Thus, PCR-dipstick DNA chromatography using the same plaque sample enabled simultaneous profiling of multiple species at species level and facilitated discrimination between anticipated different microflora, making this technique a promising chair-side microbiota profiling method.The ecological plaque hypothesis (18) has revolutionized our view of the etiology of the most prevalent oral diseases, including caries and periodontal diseases. Contrary to the classical medical model, which ascribes an infection to a single microbial pathogene, the new hypothesis suggests that a dysbiosis of the resident oral microflora, driven by an ecological perturbation, leads to plaque-mediated disease. During the transition from oral health to disease state, a dynamic interaction between the resident microflora and the habitat has been evidenced, with numerous bacterial species being implicated (2,3,22,31). A substantial change of the environmental determinants could disrupt the homeostasis of the resident microflora, thereby inducing the prolifera-

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“…A limitation of DGGE is that sequence differences greater than 1 base pair may fail to separate on a denaturing gel because of similarities in nucleotide proportions that result in identical denaturing characteristics of 2 different sequences. 12 Therefore, excision and sequencing is necessary to confirm the identification of species present within an individual band.…”

Section: S Rrna Gene Sequencingmentioning

confidence: 99%

Unravelling Mystery of Biofilm

Bhullar¹

2017

JMSCR

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Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque

Cited by 54 publications

References 26 publications

Microbial contamination of laboratory constructed removable orthodontic appliances

Microbial contamination of laboratory constructed removable orthodontic appliances

<b>PCR-dipstick DNA chromatography for profiling of a subgroup of cariesassociated bacterial species in plaque from healthy coronal surfaces and periodontal </b><b>pockets </b>

Unravelling Mystery of Biofilm

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