Quantification of Streptococcus mutans by qPCR
Purpose: To develop a SYBR® Green qPCR method for quantifying Streptococcus mutans (SM) in salivary samples.
Methods: SM specific primers were derived from the DNA sequence of AP-PCR amplification products (unpublished). Serial dilutions of overnight mutans streptococci (MS) cultures were blotted on FTA cards (Whatman Bioscience Ltd.) as standards for qPCR. Sixty clinical saliva and swab samples, from children aged 2-10 with or without caries, enumerated for SM by traditional culturing were archived on FTA cards for SM quantification by SYBR® Green qPCR. The specificity of qPCR was analyzed by product melting curves. Correlation of SM quantification by qPCR and culture was analyzed using Pearson correlation test. The correlation of MS quantification by culture, or SM quantification by qPCR, and caries scores were analyzed by Pearson correlation test.
Results: A total of 60 clinical samples (20 saliva and 40 swab samples) were enumerated by traditional culturing, and analyzed by qPCR. The 20 saliva samples were from caries-active (high caries risk) children, and the 40 swab samples were from both caries-active and caries-free children. The SYBR® Green qPCR method provided high efficiency and good specificity for SM detection in salivary samples archived on FTA cards. qPCR showed good reproducibility with a mean variance (CV) = 0.13 for qPCR samples with log10 SM > 3 CFU/ml. SM quantification by qPCR was significantly correlated (Pearson's correlation) with MS culture enumeration for all clinical samples, (r=0.51, P<0.001), saliva samples (r=0.52, P<0.05), and swab samples (r=0.51, P<0.001).
In addition for swab samples, qPCR and culture results were significantly correlated with caries status (DS, r=0.32, P<0.05 for qPCR; DMFS and DS r=0.51, P<0.001 and r=0.44, P<0.01, respectively for culture).
Conclusions: qPCR quantification of SM showed good correlation with traditional culture results. It offers a fast and convenient quantification method for SM in clinical samples and could facilitate SM quantification for private practitioners. Development of qPCR techniques to quantify other cariogenic bacteria is needed for thorough evaluation of bacterial challenge.