实时荧光PCR方法检测结核分枝杆菌mRNA的研究

目的建立检测结核分枝杆菌mRNA表达水平的实时荧光PCR反应体系,快速鉴定结核分枝杆菌。方法设计合成结核分枝杆菌mRNA相应引物和探针,探针标记以JOE为报告基团,BHQ1为猝灭基团。优化反应条件,建立结核分枝杆菌mRNA的实时荧光PCR检测方法。通过检测22份肺结核患者痰液、10份健康无症状人群痰液、结核分枝杆菌标准菌株和偶然分枝杆菌菌株培养菌落,检验方法的特异性、重复性和灵敏度。结果肺结核患者痰液检测均出现典型扩增曲线,CT均值31.35,标准差4.31。健康无症状人群痰液及偶然分枝杆菌未出现扩展曲线。对痰液标本及稀释RNA重复检测的变异系数在2.54%以下,检测CT值与痰涂片抗酸染色分枝杆菌的数量呈良好相关性。结论建立了一种快速实时检测结核分枝杆菌mRNA基因的荧光PCR方法,具有良好的敏感性和特异性。

Detection method of sputum Mycobacterium tuberculosis messenger RNA with real-time PCR

Objective To establish a real-time RT-PCR method for quickly identifying Mycobacterium tuberculosis messenger RNA（mRNA）. Methods Mycobacterium tuberculosis mRNA primers and probe were designed, and probe was labelled by JOE-BHQ1. A total of 22 samples of TB patients＇ sputum,10 samples of healthy symptomless peoples＇ sputum, M. tuberculosis ATCC 27294, M. fortuitum ATCC 6841 were tested to evaluate the specificity,reproducibility and sensitivity of the method. Results All TB patients＇ sputum were tested positive for TB mRNA by real-time PCR method with CT average value 31.35（SD 4.31）. M.tuberculosis ATCC 27294 was also positive, while10 healthy symptomless peoples＇ sputum and M. fortuitum ATCC 6841 were negative. TB mRNA reproductivity testing showed CT＇s CV were about 2.54%. Conclusion This real-time PCR method could detect TB mRNA with appropriate sensitivity and specificity.