反向斑点杂交法检测流感嗜血杆菌核酸

Detection of Haemophilus influenzae by reverse dot-blot hybridization

目的 探讨早期、快速检测流感嗜血杆菌的方法。方法 应用流感嗜血杆菌编码外膜蛋白特异的P6基因设计引物 ,通过聚合酶链反应合成特异探针 ,采用反向斑点杂交法检测生物素标记DNA ,并应用于痰标本的检测。结果 只有流感嗜血杆菌扩增出 35 1bp的DNA片段 ,该探针能检测出 10ng的细菌DNA ,与其他细菌、病毒、真菌无交叉反应。该方法和培养法分别检测 5 0份痰标本 ,两者的阳性率分别为 30 %和 2 2 %。结论 该方法快速、特异 。

Objective To explore a rapid method for early detection of Haemophilus in fluenzae.Methods A pair of primers was designed according to the P6 gene encoding outer membrane protein of Haemophilus influenzae.The specific probes were synthesized by polymerase chain reaction.Bacterium DNA labeled with biotin in sputum specimens was detected by reverse Dot-blot hybridization.Results Only a 351 bp DNA fragment was amplified from haemophilus influenzae.The probes can detect as low as 10 ng of bacterium DNA.No cross hybridization with the DNA of other bacteria, viruses and fungi occurred.Fifty sputum specimens were identified by the developed method and culture simultaneously and the positive rates were 30% and 22% respectively.Conclusion The method is of value in rapid and specific identification of haemophilus influenzae.