变形链球菌(血清型C)临床分离株AP-PCR基因分型

Typing of Streptococcus mutans (serotype C) by arbitrarily primed polymerase chain reaction

目的 探讨变形链球菌 (血清型C)菌珠基因型与龋发生的关系。方法 用Chelex法提取细菌染色体DNA ,经多次实验自行确立AP PCR最佳反应条件 ,并对 2 78株来自不同龋敏感个体的临床分离株进行AP PCR基因型分析。结果 2 78株变链菌临床分离株共区分出 10 5种基因型 ,无龋个体 84 2 1%携带 1种基因型 ,而高龋患者 95 %携带 2种或 2种以上基因型。结论 变链菌菌株间存在明显的遗传多态性 。

Objective To study the relationship between genetic diversity within Streptococcus mutan s (C) and dental caries. Methods One reference strain (GS5) w as used to establish the suitability of primers and the initial AP-PCR conditio n s. Isolates of Streptococcus mutans (C) were obtained from 19 caries-free a nd 40 caries-active patients, and were originally isolated on mitis-salivarius -bacitracin agar. Totally 278 isolates were biochemically and serologically con firmed 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 re action. All amplification reactions were conducted in a volume of 20 μl contain ing 2 μl of 10×reaction buffer, 200 μmol/L each of dATP, dCTP, dGTP and dTTP and 25 units of Taq DNA polymerase. We varied the concentrations of MgCl 2 (15 ～45 mmol/L), template DNA (2～40 ng), and primer (03～05 μmol/L) to dete rmine th e optical conditions for AP-PCR. The temperature profile in a thermocycler was 35 cycles as follows: 94℃ for 1 min, at 36℃ for 2 min, at 72℃ for 2 min . The initial denaturation was at 94℃ for 5 min and the final extension at 72℃ f or 5 min. Results The MgCl 2 concerntration in 4 mmol/L and the primer concerntration in 0.5 μmol/L produced clearly distinct and reproducible finge rprin ts. We found 105 different patterns among the 278 isolates. In high-caries pers ons more than one genotype isolates of Streptococcus mutans (C) were coloniz ed. Conclusions Isolates of Streptococcus mutans (C) exist apparent genetic diversity. The genotypes of isolates might relate to difference s in caries susceptibility.