聚合酶链反应微孔板杂交法检测解脲脲原体及药敏分析

Detection of ureaplasma urealyticum by polymerase chain reaction microplate hybridization and anti Uu susceptibility test

目的 建立敏感、特异的聚合酶链反应 微孔板杂交法 (PCR MPH)检测解脲脲原体 (Uu)的感染 ,并分析药敏情况。方法 将带有生物素标记的Uu的聚合酶链反应产物结合在链霉亲和素包被的微孔板上 ,与标记地高辛的探针进行杂交后 ,通过标记碱性磷酸酶的抗地高辛抗体 (anti DIG AP)与杂交分子进行反应 ,最终经底物显色后读数 ;同时应用生物梅里埃公司支原体培养试剂盒 (IST)进行了Uu的培养及药敏试验。结果 通过优化多种试验条件建立了PCR MPH法检测Uu ,并分析了不同人群 15 8份标本 ,其中微孔板杂交法阳性 6 5份 ,培养法阳性 5 6份 ;药敏结果显示 ,敏感率原始霉素 10 0 %、交沙霉素 96 .6 %、强力霉素 89.7%、四环素 79.3%、氧氟沙星 34 .5 %、红霉素 6 .9%。结论PCR MPH法采用非放射性标记 ,具有无污染、经济、自动读数等优点 ,可以敏感、特异地检测Uu的感染 ;Uu对原始霉素、交沙霉素、强力霉素较敏感 。

Objectives To establish a sensitive and special method for the detection of Ureaplasma urealyticum(Uu) using PCR microplate hybridization (PCR MPH). Methods A primer of ureasea gene was labeled by biotin. The amplification product was captured on streptavidin coated microplates, then products were quantified by hybridization with a digoxigenin labeled internal oligonucleotide probe. After revelation with an anti digoxigenin alkaline phosphatase coupled antibody(anti DIG AP), the amount was determined by optical reading. At the same time, PCR MPH was compared with Bio Merieux Mycosplasma IST. Results A method of PCR MPH for detecting Uu DNA was established. The morbidity among three groups for detecting 158 clinical samples was analysed. 65 were detected by PCR MPH and 56 by culture.Conclusion The results showed that this assay is rapid, sensitive, specific, and accurate, and is of value in clinical therapy.