反向斑点杂交法快速检测肺炎支原体

Rapid detection of mycoplasma pneumoniae by reverse Dot blot hybridization

目的:探讨早期、快速检测肺炎支原体的方法。方法:根据肺炎支原体特异的P1基因设计引物,通过聚合酶链反应合成一段特异的162bp长链DNA探针,采用反向斑点杂交法检测生物素标记支原体DNA,并应用于痰标本的检测。结果:所合成的162bpDNA探针具有高度特异性,只和肺炎支原体杂交,与其它细菌、真菌、病毒无交叉反应,该探针最低可检测出1ng的DNA。杂交法和培养法分别检测100份痰标本,两者的阳性率分别为12%和2%。结论:该方法快速、特异,对肺炎支原体的早期诊断具有较高的应用价值。

Objective To explore a rapid method for detection of mycoplasma pneumoniase. Methods A pair of primers was designed according to the P1 adhesion gene of mycoplasma pneumoniae. A specific 162 bp DNA probe was synthesized by polymerase chain reaction. Bacterium DNA labeled with biotin and 100 sputum specimens of patients with CAP were detected by reverse Dot-blot hybridization. Results The 162 bp DNA probe synthesized was highly specific, The probe only hybridized with mycoplasma pneumoniae without cross-reaction with other bacteria, fungi or viruses. The probe can detect as low as 1ng of bacterium DNA. 100 sputum specimens were identified with the hybridization and culture method. The positive rates of them were 12% and 2% respectively. Conclusion This method is of high value in rapid and specific identification of mycoplasma pneumoniae.