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Published on: October 23, 2011
[Development of a qPCR assay for quantitative detection of Streptococcus mutans utilizing lyophilized reference
Rui Guo1, Yuefeng Wang2, Qian Liao1
1Beijing Stomatological Hospital, Capital Medical University, Beijing 100070, China.
Objective:
To establish a quantitative real-time polymerase chain reaction (qPCR) method for detection of Streptococcus mutans based on ready-to-use lyophilized reference materials (RMs) and apply it in screening of probiotics against cariogenic S. mutans.
Methods:
Primers and probes were designed targeting the gtfB gene, a specific single-copy gene associated with the core cariogenicity of S. mutans. A qPCR detection method was established and systematically validated. Ready-to-use lyophilized RMs derived from S. mutans ATCC 25175 with high uniformity and stability were prepared to construct a quantitative regression model between cycle threshold (Ct) and colony forming units (CFU). This was then utilized to accurately quantify S. mutans in multi-species cultures mixed with four strains of probiotic, respectively and thereby evaluating their inhibitory activities.
Results:
The established qPCR method exhibited excellent analytical specificity, yielding positive amplification only for 10 strains of S. mutans (Ct=14.84-16.79) with no cross-reactivity against 18 strains of microbials other than S. mutans. The limit of quantification was 2.10×103 CFU/mL, showing a high concordance rate (91.59%-108.76%) with traditional plate counting method. The prepared lyophilized RMs met the China national standard requirements for uniformity, with a mean Ct value of 17.45 (SD=0.13) across 20 individual samples, allowing for direct quantitative detection without pre-activation. Evaluation of intervention revealed that all four strains of probiotics (three for Ligilactobacillus salivarius and one for Lacticaseibacillus paracasei) exerted inhibitory effects on S. mutans with a characteristic of strain-specific. The inhibitory potency followed the order of L. salivarius BH0006> BH0004> BH0005> L. paracasei BH0013.
Conclusion:
The qPCR quantification assay based on standardized lyophilized RMs developed in this study is of high sensitivity, specificity, and accuracy. It effectively overcomes the technical bottleneck of quantifying S. mutans in multispecies cultures.
