寡发酵链球菌分子标识鉴定方法的建立

Development of an identification method for Streptococcus oligofermentans:a new species of oral streptococci with molecular markers

目的建立快速、有效鉴定寡发酵链球菌的方法,并验证其准确性。方法以9种口腔链球菌11株标准菌株的 DNA 为模板,用寡发酵链球菌16S rDNA 的特异引物和乳酸氧化酶基因的引物通过 PCR 分别扩增这两个基因的部分片段,建立用分子标识鉴定该菌的方法。并从9个无龋的志愿者口腔中取得集合牙菌斑,在加红霉素的轻唾琼脂平板中进行培养,分离到疑似寡发酵链球菌的菌落后,用已建立的方法进行鉴定。并对初步鉴定为寡发酵链球菌的分离株进行16S rDNA 序列同源性分析,以确定鉴定的准确性。结果用建立的分子标识鉴定方法,发现在11株标准菌株中只有3株寡发酵链球菌有扩增产物;用该方法鉴定为寡发酵链球菌的来自无龋志愿者牙菌斑的分离株,经16S rDNA 序列同源性分析证实确实为寡发酵链球菌。结论用寡发酵链球菌16S rDNA 特异性引物和乳酸氧化酶基因引物进行两步 PCR 来鉴定寡发酵链球菌是准确可靠的。

Objective To establish a quick and reliable method to identify Streptococcus oligofermentans, a new species of oral streptococci. Methods With two-step PCR, a pair of the 16S rDNA- specific primers of Streptococcus oligofermentans and a pair of primers of lactate oxidase gene （lox） were used to amplify the gene fragments from the genomic DNAs of 11 strains consisting of 9 species of the pure culture of oral streptococci. Pooled plaque samples from 9 caries-free volunteers were cultured on a selective medium of MSA with erthromycin and tentative strains of Streptococcus oligofermentans were isolated. The isolates were further identified by the two-step PCR and finally confirmed by 16S rDNA sequence analysis. Results With the two-step PCR, the two gene fragments were only amplified from the three identified strains of Streptococcus oligofermentaus, but not the rest of 8 strains of oral streptococci. Isolates from the dental plaque of caries-free volunteers were identified as Streptococcus oligofermentaus by PCR and then further confirmed by 16S rDNA sequence homology analysis. Condusions Streptococcus oligofermentaus could be identified by the two-step PCR approach with the specific 16S rDNA primers and lactate oxidase gene primers.