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[Typing of Streptococcus mutans (serotype C) by arbitrarily primed polymerase chain reaction]
1Department of Operative Dentistry and Endodontics, Hospital for Stomatology, Fujian University of Medical Sciences, Fuzhou 350001, China.
Objective:
To study the relationship between genetic diversity within Streptococcus mutans (C), and dental caries.
Methods:
One reference strain (GS5) was used to establish the suitability of primers and the initial AP-PCR conditions. Isolates of Streptococcus mutans (C) were obtained from 19 caries-free and 40 caries-active patients, and were originally isolated on mitis-salivarius-bacitracin agar. Totally 278 isolates were biochemically and serologically confirmed as Streptococcus mutans (C). Chromosomal DNA was extracted from those isolates by Chelex. One random primer, OPA-05 was used as a template in PCR reaction. All amplification reactions were conducted in a volume of 20 microliters containing 2 microliters of 10 x reaction buffer, 200 mumol/L each of dATP, dCTP, dGTP and dTTP and 2.5 units of Taq DNA polymerase. We varied the concentrations of MgCl2 (1.5-4.5 mmol/L), template DNA (2-40 ng), and primer (0.3-0.5 mumol/L) to determine the optical conditions for AP-PCR. The temperature profile in a thermocycler was 35 cycles as follows: 94 degrees C for 1 min, at 36 degrees C for 2 min, at 72 degrees C for 2 min. The initial denaturation was at 94 degrees C for 5 min and the final extension at 72 degrees C for 5 min.
Results:
The MgCl2 concentration in 4 mmol/L and the primer concentration in 0.5 mumol/L produced clearly distinct and reproducible fingerprints. We found 105 different patterns among the 278 isolates. In high-caries persons more than one genotype isolates of Streptococcus mutans (C) were colonized.
Conclusions:
Isolates of Streptococcus mutans (C) exist apparent genetic diversity. The genotypes of isolates might relate to differences in caries susceptibility.