DNA探针杂交快速检测肺炎链球菌和流感嗜血杆菌

Rapid detection of Streptococcus pneumoniae and Haemophilus influenzae with DNA probes

目的建立一种早期快速检测肺炎链球菌和流感嗜血杆菌的分子生物学方法。方法采用聚合酶链反应(PCR)合成肺炎链球菌和流感嗜血杆菌特异的DNA探针和细菌16SrRNA的通用探针,并用生物素标记DNA探针,分别与细菌、病毒和真菌DNA杂交。并将该方法评价临床样本的检测。结果聚合酶链反应分别扩增出263、351、370bp三种DNA探针,肺炎链球菌和流感嗜血杆菌只与其对应的探针杂交,细菌通用探针和病毒、真菌间无交叉反应。该法可检测出1ng细菌DNA,100份痰标本利用该方法检测出11份肺炎链球菌和8份流感嗜血杆菌,其阳性率高于痰培养。结论该方法简便、快速、特异,具有较高的应用价值,可应用于临床检测。

Objective To establish a method for rapid molecular detection of Streptococcus pneumoniae and Haemophilus influenzae in the early stage of infection. Methods Specific DNA probes for Streptococcus pneumoniae and Haemophilus influenzae and 16 S rRNA universal probe of bacteria were synthesized by polymerase chain reaction （PCR） and labeled with biotin. The DNA of the bacteria, virus and fungi were hybridized with these probes respectively prior to application for examination of the clinical samples. Results The DNA probes of 263, 351, and 370 bp were amplified by PCR. Streptococcus pneumoniae and Haemophilus influenzae reacted only with their corresponding probes, and no cross reaction of the bacterial universal probe with virus and fungi was noted. The method could detect bacterial DNA in as small amount as 1 ng. Of the 100 sputum specimens, 11 were found to be positive for Streptococcus pneumoniae and 8 for Haemophilus influenzae, with a positivity rate greater than that by sputum culture. Conclusion DNA probe detection is simple, rapid, and specific for clinical examination of Streptococcus pneumoniae and Haemophilus influenzae.