Design and validation of a quantitative polymerase chain reaction test for the identification and quantification of uncultivable bacteria associated with periodontitis

Autores organización

• Yormaris Castillo Romero

  Autor

• 

  Nathaly Andrea Delgadillo Salgado

  Autor

• Yineth Neuta Poveda

  Autor

• 

  Gloria Ines Lafaurie Villamil

  Autor

• 

  Diana Marcela Castillo Perdomo

  Autor

Autores

• Iniesta M
• Sanz M
• Herrera D
• Pianeta R

Grupos de investigación

• Unidad de Investigación Básica Oral UIBO

  

  Investigador

  Gloria Ines Lafaurie Villamil

Resumen

Objective: This study aimed to standardize a quantitative polymerase chain reaction (qPCR)-based test to identify and quantify the uncultivable bacteria associated with periodontitis. Methods: The standardization of qPCR, the curves for the quantification of Eubacterium saphenum, Eubacterium brachy, Desulfobulbus oralis, and Filifactor alocis were developed by cloning the 16 S rRNA target gene fragment, using the GEMTEasy vector. The qPCRs were validated in 55 subgingival biofilm clinical samples, from different stages of periodontitis and from periodontally healthy/gingivitis individuals, which were previously evaluated by next-generation sequencing (NGS). The results obtained by the two methods were compared by the concordance of Cohen's Kappa index, and sensitivity, specificity, receiver operating characteristic (ROC) curve, and predictive values were established. Results: obtained by the two methods were compared using the concordance of Cohen's Kappa index, and sensitivity, specificity, predictive values, and ROC curves were generated. The qPCR test was standardized with efficiencies between 90% and 100% and R2: 0.997–0.999. Concordance between the qPCR and NSG was moderate to F. alocis (agreement 78.2%; kappa 0.56, p < 0.05) and fair to the other microorganisms (agreement 67.27%-72.73; kappa 0.37–0.38, p < 0.05). qPCR exhibited a high sensitivity (82.2–100%) and specificity (100%) for E. brachy, E. saphenum, and F. alocis. Sensitivity was lower to D. oralis. Conversely, qPCR demonstrated higher sensitivity to E. saphenum than NSG (100 vs. 68.1). Conclusions: The uncultivable microorganisms associated with periodontitis, D. oralis, E. brachy, E. saphenum, and F. alocis can be detected and quantified with the newly developed and validates qPCR test. © 2023

Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.

Keywords

• Gingivitis; Humans; Periodontitis; Polymerase Chain Reaction; Porphyromonas gingivalis; genetics; gingivitis; human; microbiology; periodontitis; polymerase chain reaction; Porphyromonas gingivalis; procedures