Quantification of Bacteria in Oral Samples by Competitive Polymerase Chain Reaction

Abstract

Information about the total amount of bacteria in oral samples contributes to assessment of an individual's risk of contracting dental caries or developing periodontitis and the prediction of that individual's clinical course. Since existing techniques are often time-consuming and expensive, it seemed attractive to look for alternative methods for the quantification of eubacteria. With their high specificity and sensitivity, polymerase chain-reaction (PCR) techniques have the potential of supplying fast and reliable results. We developed a method of competitive PCR for the quantification of eubacteria. We designed forward and reverse PCR primers which bind to highly conserved sequences of the bacterial 16S rRNA gene. A homologous competitor was synthesized with Escherichia coli 16S rDNA as a template, with the reverse primer and a hybrid primer which binds 67 bases downstream to the forward primer and carries the forward primer sequence at its 5' end. Specificity controls with 30 different bacterial species, 5 Archaea, 3 fungi, human astrocytoma cells, and rat hepatoblastoma cells were carried out. Results were positive for all eubacteria and negative for all other cells tested. Calibration curves were obtained by co-amplification of known amounts of E. coli cells in the presence of the homologous competitor. The developed method was successfully applied to assessment of the accumulation of bacteria during an oral hygiene cessation experiment. The competitive PCR method proved to be a reliable and fast method for the quantification of bacterial DNA and cultured eubacteria, as well as of bacteria in biological samples. It may find further applications not only in periodontology and cariology but also in other fields of medical microbiology.

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

“…A majority of these studies, however, report the use of more than a single set of primers to detect the bacteria of interest. Other techniques, such as competitive PCR (Blok et al, 1997 ;Rupf et al, 1999), are labour-intensive and require the analysis of results from multiple reactions for each test sample. In contrast, realtime PCR, such as the TaqMan system developed by Applied Biosystems, relies on the release and detection of a fluorescent signal following the cleavage of a fluorescent labelled probe by the 5h-exonuclease activity of Taq polymerase.…”

Técnica De Reacción De Polimerasa en Cadena (Qpcr) en Tiempo Real Para La Identificación Y Cuantificación De Streptococcus Mutans en Saliva Y Biopelícula Dentaria De Niños

“…The analysis of these parameters allows validating the results thanks to the sensitivity and accuracy of this technique, as previously published, [24][25][26][27] with similar methodology. It is therefore possible to claim DISCUSIÓN Los resultados obtenidos de la amplificación mediante qPCR mostraron un alto porcentaje de eficiencia y un razonable delta entre cada ciclo.…”

“…It is therefore possible to claim DISCUSIÓN Los resultados obtenidos de la amplificación mediante qPCR mostraron un alto porcentaje de eficiencia y un razonable delta entre cada ciclo. El análisis de estos parámetros permite validar los resultados obtenidos, basados en la sensibilidad y exactitud de la técnica, tal como han sido publicados previamente, [24][25][26][27] que la metodología presentada identifica y cuantifica la especie Streptococcus mutans en muestras de saliva y biopelícula dental.…”

“…24,[25][26][27][28] Esta técnica basada en fluorescencia, 15, 29 a diferencia de la cuantificación por cultivo, no requiere personal entrenado para detectar colonias compatibles con S. mutans, las cuales adicionalmente necesitan confirmación a nivel de especie, que puede ser mediante pruebas bioquímicas o moleculares, considerando que este laborioso proceso de cultivo, aislamiento e identificación demanda mayor tiempo y recursos, y comparada con la cuantificación por qPCR resulta más rápida, económica, precisa y sensible; su único inconveniente consiste en que no discrimina la cuantificación entre células vivas y muertas. 24,[30][31] Por otra parte, esta técnica mejorada de la PCR de tiempo final o convencional, que es un ensayo cualitativo, permite realizar mediciones cualitativas y cuantitativas de genes específicos en una muestra de ácido nucleico.…”

“…[21][22][23] In the past, quantification was conducted through traditional cultivation, but technological advances have demonstrated that real-time polymerase chain reaction (PCR) can be a sensitive, quick methodology, revolutionizing the field of quantification methods. 24,[25][26][27][28] Unlike quantification by cultivation, this fluorescence-based technique 15,29 does not require trained staff to detect colonies compatible with S. mutans, which needs an additionally step of species confirmation, which can be done through biochemical or molecular tests; this arduous process of cultivation, isolation, and identification requires more time and resources, and compared against quantification by qPCR is faster, inexpensive, accurate, and sensitive; its only drawback is that it does not discriminate quantification between living and dead cells. 24,[30][31] On the other hand, this improved technique of conventional PCR, which is a qualitative test, allows conducting qualitative and quantitative measurements of specific genes in a sample of nucleic acid.…”