Highly accurate measurement of the relative abundance of oral pathogenic bacteria using colony-forming unit-based qPCR.

Abstract

Purpose: Quantitative polymerase chain reaction (qPCR) has recently been employed to measure the number of bacterial cells by quantifying their DNA fragments. However, this method can yield inaccurate bacterial cell counts because the number of DNA fragments varies among different bacterial species. To resolve this issue, we developed a novel optimized qPCR method to quantify bacterial colony-forming units (CFUs), thereby ensuring a highly accurate count of bacterial cells.

Methods: To establish a new qPCR method for quantifying 6 oral bacteria namely, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia, Fusobacterium nucleatum, and Streptococcus mutans, the most appropriate primer-probe sets were selected based on sensitivity and specificity. To optimize the qPCR for predicting bacterial CFUs, standard curves were produced by plotting bacterial CFU against Ct values. To validate the accuracy of the predicted CFU values, a spiking study was conducted to calculate the recovery rates of the predicted CFUs to the true CFUs. To evaluate the reliability of the predicted CFU values, the consistency between the optimized qPCR method and shotgun metagenome sequencing (SMS) was assessed by comparing the relative abundance of the bacterial composition.

Results: For each bacterium, the selected primer-probe set amplified serial-diluted standard templates indicative of bacterial CFUs. The resultant Ct values and the corresponding bacterial CFU values were used to construct a standard curve, the linearity of which was determined by a coefficient of determination (r²) >0.99. The accuracy of the predicted CFU values was validated by recovery rates ranging from 95.1% to 106.8%. The reliability of the predicted CFUs was reflected by the consistency between the optimized qPCR and SMS, as demonstrated by a Spearman rank correlation coefficient (ρ) value of 1 for all 6 bacteria.

Conclusions: The CFU-based qPCR quantification method provides highly accurate and reliable quantitation of oral pathogenic bacteria.