利用PCR技术检测537份新疆结核病人痰标本中的结核分枝杆菌

THE DETECTION OF 537 SPUTUM SAMPLES FROM XINJIANG TB INSTITUTE BY USING PCR TECHNIQUE

目的 提高聚合酶链反应在检测人结核分枝杆菌中的特异性和敏感性。方法 在聚合酶链反应中对 4种DNA片段即结核杆菌特异插入序列IS6 110 ,IS10 81,和 16SrRNA ,6 5kDa摸板进行了比较 ;为获取较多的细菌DNA ,临床痰标本处理采用了国际标准化方法主要包括用一定量的N -乙酰 -L半胱胺酸和氢氧化钠处理痰标本 ;改进了RCR混和液的成份即添加了甘油 ,dUTP -尿嘧啶糖基化酶。结果 选择IS6 110作为结核杆菌特异性摸板用于检测细菌DNA ,制备出了 6批人结核杆菌PCR检测试剂盒 ,在对来自新疆结核病研究所的 5 37份痰标本的检测中发现 ,该试剂盒检测的特异性为 6 5 .97% ,敏感性为 93 .5 3 %。结论改良TB -PCR试剂盒显示有较高的敏感性 ,特异性还有待于进一步完善 ,该试剂盒在临床诊断中有一定的价值。

Aim To improve the specificity and sensitivity of polymerase chain reaction(PCR)technique for detecting of DNA of M.tuberculosis.Method Four frangments of IS6110,IS1081,16SrRNA,65kDa were compared as template of PCR in this experiment;international standardized specimen-processing method for improved recovery of mycobacteria and the methods were included in sputum treating with acetyl-L-cysteine and sodium hydroxide (NALC-NaOH);to improve the PCR mixture including in adding glycerol,dUTP,Uracil-N-glycosylase.Result The IS6110 was chosen as template for detecting DNA of M.TB.The six batch of TB-PCR KIT were prepared.The 537 samples from Xinjiang TB institute were tested with TB-PCR KIT,the specificity was 65.97% and sensitivity was 93.53%.Conclusion TB-PCR KIT method shows higher sensitivity and specificity and will be improved in future.it is of clinical value in diagnosis of TB.