Absolute quantitation of oral bacteria involved in oral cancer by real-time PCR

Abstract

India carries approximately 34% of the global oral cancer burden. Along with tobacco chewing habit, there are other factors that are being studied globally like the involvement of bacteria in oral cancer. NGS studies have identified few bacterial species and the changes in their abundance in healthy and diseased populations based on 16S rRNA metagenomics. The present study shows a method for absolute bacterial quantification from oral cavity rinse samples. The method is a real-time qPCR assay and based on the fact that certain genes are present in one copy per cell (rpoB gene) and we can correlate the copy numbers of these genes with cell numbers in a sample. This method is more accurate than the NGS 16S rRNA gene-based approach which is multicopy gene. Linear correlation between qPCR assay and cell numbers of a model system was established. Consequently, the assay was performed on oral rinse samples of oral cancer/tobacco chewers and healthy subjects to quantitate significant oral bacterial species. The obtained bacterial quantification correlated well with the previous reports. The developed qPCR method is an efficient, faster and resource-friendly method and can be used to quantify bacterial population in cancer/diseased subjects, and can have application in determining the susceptibility of an individual towards a specific disease.