反向斑点杂交快速检测肺炎链球菌

目的:建立一种利用DNA探针快速检测肺炎链球菌的反向斑点杂交方法.方法:在肺炎链球菌特异的自溶素基因序列内设计引物,聚合酶链反应合成1段263 bp的DNA探针,然后用生物素标记的细菌DNA与该探针行反向斑点杂交,并将该方法应用于痰样本的检测.结果:所合成的探针具有高度特异性,与其他细菌间无交叉反应,该方法能检测出10 ng细菌DNA.50份痰标本肺炎链球菌培养阳性者8份,杂交阳性者12份,PCR阳性者18份.结论:反向斑点杂交具有快速、敏感、特异的优点,对肺炎链球菌的快速诊断有重要意义.

Rapid detection of Streptococcus pneumoniae by reverse dot-blot hybridization

Objective: To establish a method of reverse dot blot hybridization for rapid detection of Streptococcus pneumoniae with DNA probe. Methods: A pair of primers was designed according to the lyt gene of Streptococcus pneumoniae. The DNA probe of 263 bp was synthesized by polymerase chain reaction. The bacterium DNA labeled with biotin was hybridized with the DNA probe. The same method was applied for examination of sputum samples. Results: The probe was specific and no cross hybridization with other bacteria occurred. The method could detect bacterial DNA as low as 10 ng. Eight isolates of Streptococcus pneumoniae were cultured from 50 suptum samples, of which 12 were positive by hybridization and 18 were positive by PCR. Conclusion: Reverse dot blot hybridization is a rapid, sensitive and specific method for rapid detection of Streptococcus pneumoniae.