Rapid isolation of chromosomal DNA from oral Streptococci and polymerase chain reaction oriented restriction fragment-length polymorphism analysis for genetic heterogeneity
Rapid isolation of chromosomal DNA from oral Streptococci and polymerase chain reaction oriented restriction fragment-length polymorphism analysis for genetic heterogeneity
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DOI：
10.1111/j.1399-302x.1998.tb00744.x
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发表时间：
1998-02-01
影响因子：
--
通讯作者：
Abiko, Y
中科院分区：
文献类型：
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作者：
Shiroza, T;Shinozaki, N;Abiko, Y
我们对已发表的革兰氏阳性菌裂解方法进行了广泛的修改，仅从1ml的口腔链球菌过夜培养中分离出染色体DNA。细胞与溶菌酶和rna酶A在聚乙二醇存在下孵育。离心后，用十二烷基硫酸钠和蛋白酶k裂解细胞，乙醇沉淀后，加入十二烷基硫酸钠溶液，65℃孵育使颗粒完全分散，用苯酚和氯仿萃取纯化染色体。用聚合酶链反应（PCR）方法扩增了变形链球菌和sobrinus链球菌染色体核糖体RNA （rrn）操纵子和糖基转移酶基因对应的两个区域。采用限制性片段长度多态性（PCR-RFLP）评价遗传异质性。PCR-RFLP分析很容易让我们确定每个菌株的亚型，这表明本文提出的策略将为在分子水平上验证流行病学研究提供有用的工具。
We have extensively modified the published method for the lysis of gram-positive bacteria to isolate chromosomal DNA from only 1 ml of oral streptococcal overnight culture. Cells were incubated with lysozyme and RNase A in the presence of polyethylene glycol. After centrifugation, cells were lysed with sodium dodecyl sulfate and proteinase K. Following ethanol precipitation, sodium dodecyl sulfate solution was added to the residue, and the pellet was completely dispersed by incubating at 65 degrees C. The chromosome was purified by extraction over phenol and chloroform. Two regions corresponding to the ribosomal RNA (rrn) operon and the glucosyltransferase gene were amplified using the chromosome from Streptococcus mutans and Streptococcus sobrinus by polymerase chain reaction (PCR). Genetic heterogeneity was assessed by restriction fragment-length polymorphism (PCR-RFLP). The PCR-RFLP analysis readily allowed us to subtype each strain, suggesting that the strategy presented here will provide a useful tool to verify epidemiological studies at the molecular level.