微孔杂交技术诊断肺外结核

Extrapulmonary tuberculosis diagnosed by microcell plate hybridization assay

目的 将聚合酶链反应 (PCR)、核酸杂交、酶联免疫吸附三种技术有机结合 ,从分子水平检测结核杆菌 ,为临床诊断肺外结核提供病原学依据。方法 使用生物素标记的特异性引物扩增结核杆菌 (TB)DNA ,带有生物素的产物与包被在微孔板内的靶基因杂交 ,加入酶标链毒亲和素与生物素结合 ,最后加辣根过氧化物酶显色 ,2MH2 SO4 终止反应。根据酶标仪检测各孔吸光度判断结果。结果通过对临床高度怀疑为肺外结核的 4 0 8份标本的检测 ,PCR 微孔板杂交法的检出率为 4 3.6% ,培养法的检出率为 2 5.9%直接涂片抗酸染色为 6.4 %。结论 PCR杂交法灵敏度高 ,特异性强 ,有助于临床准确、快速诊断肺外结核。

Objective Mycobacterium tuberculosis was detected by the method combining polymerase chain reaction(PCR),hybridization,ELISA,so that the founding of the pathogen can be thought as the diagnostic evidence of tuberculosis.Methods The Mycobacterium tuberculosis DNA was amplified by the specific primers labeled with biotin at 5'end,PCR products with biotin were hybridized with target DNA precoated in the microcell,then the hybrid can be detected by strepavidin labeled with HRP,the next process was similar to ELISA.ResultsAmong the 408 samples which may be contain Mycobacterium tuberculosis,the positive rate were 43.6 percent by PCR hybridization method,25.9 percent by culture,6.4 percent by direct microscopy(Ziehl-Neelsen staining)Conclusion PCR hybridization assay was sensitive specific and fast method for the dignosis of extrapulmonary.