结核分支杆菌扩增体系的建立、优化及评价

The establishment,optimization and evaluation of PCR system for the detection of bacillus tuberulosis

目的建立并优化结核分支杆菌PCR反应扩增体系,并对所建立的体系进行评价。方法根据GenBank找到结核分支杆菌基因序列,在Primer Premier V5.0软件上设计结核分支杆菌特异性引物。并对反应体系中MgCl2、引物、dNTP浓度以及引物退火温度优化后建立结核分支杆菌PCR反应扩增体系。通过对TB标准株、TB临床分离株和临床阳性标本扩增产物的直接测序、灵敏性试验以及特异性试验对PCR反应体系进行综合评估。结果TBPCR反应体系阳性扩增结果为在琼脂糖凝胶电泳上出现245bp的条带。MgCl2、引物以及dNTP在TBPCR体系中优化后最佳终浓度分别为:4mmol/L、0.04μmol/L以及0.3mmol/L。TBPCR体系的退火温度优化后为55℃。TB标准株及TB临床分离株和临床阳性标本扩增产物的直接测序结果与GenBank中公布的序列完全一致。TBPCR反应体系对其它细菌无交叉反应。检测限度为10拷贝/μl的DNA。结论TBPCR反应体系能够快速对TB标准株、TB临床分离株及临床标本进行结核分支杆菌的鉴别。

Objective To establish and opfimize the PCR system, and to evaluate this system. Methods The gene sequence of Bacillus Tuberculosis was found in GenBank, and its specific primer wad designed through Primer Premier V5.0. The concentratons of MgCl2, primers and dNTP were optimized, and the renaturation temperature was also optimized, so as to establish the optimized system. The system was also evaluated by the sequencing of the product of TB standard strain, TB clinic strain and clinic positive specimen,and evaluated by specificity assay. Results The TB PCR positive result is the appearance of 245bp band in the agarose gel electrophoresis. The optimized concentrations of MgCl2, primers and dNTP were 4mmol/L, 0.04μmol/L and 0.3 mmol/L. The optimized renaturation temperature was 55℃ .The sequencing results of the product of TB standard strain, TB clinic strain and clinic positive specimen were the same as the GenBank. TB PCR system. has no cress reaction with other bacteria. Its detection limit was 10copy/μl DNA. Conclusion TB PCR system can discriminate the TB standard strain, TB clinic stain and clinic positive specimen of TB quickly.