【摘要】 目的运用实时定量PCR(quantitative real-time,qRT-PCR)检测结核分枝杆菌蛋白Ag85B、38kDa及MPT64编码基因的表达水平,探讨结核分枝杆菌耐药菌株和敏感株,北京基因型和非北京基因型的蛋白抗原在转录水平的差异性及qRT-PCR诊断活菌的价值。方法根据Genbank提供的序列,设计目的基因fbpB、psts1、mpt64及内参基因Sigа的特异性引物,将扩增片段克隆入载体pMD18-T simple,重组质粒经纯化及倍比稀释,应用SYBR GreenⅠ荧光染料建立检测fbpB、psts1、mpt64基因表达水平的实时定量PCR方法。选用46株结核分枝杆菌临床分离株,以及2株非结核分枝杆菌。应用qRT-PCR检测上述菌株、H37Rv国际标准株fbpB、psts1、mpt64基因的表达水平。结果结核分枝杆菌活菌的扩增结果呈典型的S型曲线,而结核分枝杆菌死菌和非结核分枝杆菌呈水平直线,没有检测到荧光信号;耐药组中的mpt64基因表达水平低于敏感组及H37Rv标准株,有统计学意义(t=3.093,P<0.01;t=2.545,P<0.05),北京基因型组中的psts1基因表达水平低于非北京基因型组及H37Rv标准株。结论有统计学意义(t=2.567,P<0.05;t=2.373,P<0.05)。结论成功建立了fbpB、psts1、mpt64基因的qRT-PCR检测方法,该方法可以快速判断活菌与死菌。检测发现耐药组中的mpt64基因表达水平低于敏感组及H37Rv标准株;北京基因型组中的psts1基因表达水平低于非北京基因型组及H37Rv标准株。更多还原

【Abstract】 Objective Use quantitative real-time polymerase chain reaction method for detecting the coding gene’s expression level of Ag85B,38kDa and MPT64 in Mycobacterium tuberculosis and explore variability of their proteantigen in transcriptional level between the drug-resistant and the sensitive,between the Beijing genotype and non-Beijing genotype and the value of use quantitative real-time polymerase chain reaction diagnosing live Mycobacterium tuberculosis.Methods The specific primers of fbpB,psts1,mpt64 and Siga were designed according to GenBank database.The gene fragments were cloned to pMD18-T simple vector respectively,and recombinant plasmids were purified and quantified spectrophotometrically.We established the method of detecting the expression of fbpB、psts1、mpt64 by using SYBR Green I,and then selected 46 Mycobacterium tuberculosis strains and the Nontuberculosis mycobacteria(2) to do study.qRT-PCR method was used to detecting the gene’s expression of fbpB,psts1,mpt64 in 46 Mycobacterium tuberculosis,international standard strain H37Rv and 2 Nontuberculosis mycobacteria.Results The amplification result in live Mycobacterium tuberculosis is S curve,The death and Nontuberculosis mycobacteria can’t detecting fluorescencal signal and the curve is linear.The gene’s expression of mpt64 in drug-resistant strains is downregulated compare to sensitive strains and H37Rv,The result have statistical significance(t=3.093(P<0.01);t=2.545(P<0.05)).The gene’s expression of psts1 in Beijing genotype is downregulated compare to non-Beijing genotype and H37Rv,The result have statistical significance(t=3.093(P<0.01);t=2.545(P<0.05)).Conclusion The method of detecting the gene’s expression level of fbpB,psts1,mpt64 by real-time PCR have been estabished.This study find the gene’s expression of mpt64 in drug-resistant is downregulated compare to sensitive and H37Rv.the gene’s expression of psts1 in Beijing genotype is downregulated compare to non-Beijing genotype and H37Rv.更多还原