结核分枝杆菌双重实时RT-PCR检测方法的研究  

Measurement of Mycobacterium tuberculosis mRNA with duplex real-time PCR

作　　者：古莉冰[1] 朱玉兰[1] 王佃鹏 董瑞玲[1] 李微[1] 孙杰[1] 刘胜牙[1] 

机构地区：[1]深圳国际旅行卫生保健中心,广东深圳518033 [2]深圳市职业病防治院,广东深圳518000

出　　处：《中国热带医学》2012年第6期756-758,共3页China Tropical Medicine

摘　　要：目的建立检测结核分枝杆菌mRNA表达水平的双重实时RT-PCR反应体系,准确快速鉴定结核分枝杆菌。方法根据GenBank上已发表的结核分枝杆菌表达85B抗原的fbpB mRNA基因序列,进行对比分析,设计合成fbpB引物和探针,其中探针以CY5为报告基团,BHQ3为淬灭基团。根据WHO文献报道合成人类RNase P基因引物和探针,其中探针以FAM为报告基团,BHQ1为淬灭基团。经条件优化后,建立检测结核分枝杆菌mRNA双重实时RT-PCR方法,在同一体系内同时扩增结核分枝杆菌fbpB基因和人类RNase P基因,通过检测10份肺结核患者痰液和10份健康无症状人群痰液,检验方法的特异性、重复性和检测限性。结果肺结核患者痰液fbpB和RNase P基因均扩增阳性,健康无症状人群痰液fbpB均无扩展曲线,RNase P均扩增阳性。重复性检测显示fbpB基因重复检测的变异系数在1%以下,fbpB检测CT值与痰涂片抗酸染色分枝杆菌的数量呈良好的相关性。结论建立了一种快速双重实时RT-PCR方法能同时对结核分枝杆菌fbpB mRNA基因和人类RNase P基因进行检测,在检测fbpBmRNA基因的同时,通过检测RNase P基因监测RNA提取效果和PCR反应扩增效果。Objective To establish a real-time RT-PCR method to identify Mycobacterium tuberculosis mRNA quickly and correctly.Methods According to the gene sequences of the Mycobacterium tuberculosis fbpB mRNA from the GenBank,primers and probe were designed.RNase P primers and probe were cited from the WHO recommendation.FbpB probe was labelled by CY5-BHQ3 while RNase P probe was labelled by FAM-BHQ1.The reaction condition was optimized.A duplex real-time PCR method was established to detect fbpB and RNase P genes accordingly.The sputum of 10 TB patients and 10 healthy controls were tested to identify the specificity,reproducibility and sensitivity of the method.Results 10 sputum from the subjects was tested positive for both fbpB and RNase P genes,while sputum of the controls was only positive for RNase P gene.fbp B mRNA reproductivity was less than 1%,and fbp B mRNA level was correlated to the score of acid-fast bacilli in the sputa.Conclusion This duplex real-time PCR method could detect fbp B mRNA and RNase P RNA simutaneously.RNase P testing could monitor RNA extraction and PCR reaction effect.

关 键 词：结核分枝杆菌 fbpB MRNA RNASE P 双重实时RT-PCR 

分 类 号：R52[医药卫生—内科学]