Microbial Profiling in Pockets Related to Chronic Periodontitis Patients Using 16s RNA Metagenomics Sequencing
Condition: Periodontal Diseases
MeSH terms: Periodontitis; Periodontal Diseases; Chronic Periodontitis
Study type: Interventional

Summary: 
      The study was developed in compliance with the Helsinki Declaration on medical research
      involving the Ethics Committee at Ajman University (2017-A-DN-04). Informed consent from all
      the participants was obtained before participation in the study. The participants were
      systemically healthy with no history of antibiotics for the past three months. Patients
      recruited for the study were diagnosed as Stage II Generalized periodontitis according to the
      classification from the 2017 world workshop on the "classification of periodontal and
      peri-implant disease and conditions". Periodontal status indicating the severity of
      interdental clinical attachment loss of 3-4mm, radiographic bone loss between 15%-33% and
      with no tooth loss were included. The complexity of periodontitis with a maximum probing
      depth of ≤ 5mm with horizontal bone loss and having an extent and distribution with >30%
      teeth involved were included in the study. A total of 80 plaque samples were collected with
      the clinical characteristics of the patient comprising of age between 25-39 years, 36 females
      and 44 males. The subgingival plaque was collected using a sterile curette from the buccal
      aspect of maxillary molars and lingual aspect of mandibular incisors, in a vial containing
      200µl of Buffer CL.
    

Detailed description: 
      The collected plaque samples were then freshly prepared for the DNA isolation using ABIOpure
      TM Total DNA (version 2.0) (Cat No: M501DP100) according to the manufacturer's instruction.
      All the samples were assessed for DNA quantification using spectrophotometry and further
      quantified using the fluorometric method. This was performed using DeNovix DS-11 FX
      (DeNovix). Further to the DNA quantification, assessment of DNA integrity was resolved on a
      0.8% agarose gel with ethidium bromide. For NGS library preparation, all the samples
      underwent PCR amplification of the 16S rRNA gene in the isolated bacterial DNA. The primers
      used had targeted in the V3-V4 region of the 16S rRNA gene. The full length primer sequences,
      using standard IUPAC nucleotide nomenclature, to follow the protocol targeting this region
      are:

      16SAmpliconPCRForwardPrimer=5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
      16SAmpliconPCRReversePrimer=5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC Gel
      electrophoresis method was used for the size selection and the bioinformatics pipeline used
      for processing microbiome 16S sequence data was QIIME
    

Intervention name(s): 16s RNA Metagenomics sequencing
Intervention type(s): Genetic
Intervention description(s): The subgingival plaque was collected using a sterile curette from the buccal aspect of maxillary molars and lingual aspect of mandibular incisors, in a vial containing 200µl of Buffer CL.
Other intervention name(s): DNA isolation
Allocation: Randomized
Intervention model: Single Group Assignment
Intervention model description: . Patients recruited for the study were diagnosed as Stage II Generalized periodontitis according to the classification from the 2017 world workshop on the "classification of periodontal and peri-implant disease and conditions". Periodontal status indicating the severity of interdental clinical attachment loss of 3-4mm, radiographic bone loss between 15%-33% and with no tooth loss were included. The complexity of periodontitis with a maximum probing depth of ≤ 5mm with horizontal bone loss and having an extent and distribution with >30% teeth involved were included in the study.
Masking: Single (Investigator)
Primary purpose: Health Services Research
Keywords: metagenomics; 16S; Periodontitis, Adult