2种核酸扩增荧光方法用于临床痰标本的结核分枝杆菌检测

Detection of Mycobacterium tuberculosis in clinical sputum specimens by two fluorescent nucleic acid amplification methods

目的评价核酸恒温扩增实时荧光法与荧光定量PCR法检测临床痰标本结核分枝杆菌的效果。方法收集疑似结核病患者的痰标本,分别进行痰标本染色显微镜检查、分离培养菌株鉴定、核酸恒温扩增实时荧光法及荧光定量PCR检测,以培养为金标准获得2种方法的灵敏度和特异性,并比较2种方法的检测效能。结果收集322例疑似结核病患者样本,经培养鉴定91例标本分离出结核分枝杆菌菌株,阳性率为28.26%,痰样本显微镜检出阳性56例,阳性率为17.39%,核酸恒温扩增实时荧光法检出阳性112例,阳性率为34.78%,荧光定量PCR检出阳性118例,阳性率为36.65%;以培养鉴定为金标准,核酸恒温扩增实时荧光法和荧光定量PCR灵敏度分别为84.62%和86.81%,特异度分别为84.85%和83.12%;2种方法总一致率为93.17%。结论核酸恒温扩增实时荧光法和荧光定量PCR检测疑似肺结核病患者痰样本的结核分枝杆菌准确快速,能用于临床结核病诊断;而核酸恒温扩增实时荧光法更快速、简便,更适宜基层实验室使用。

Objective To evaluate the effects of real-time fluorescence and fluorescence quantitative PCR for detecting Mycobacterium tuberculosis in clinical sputum samples.Methods Sputum samples from suspected tuberculosis patients were collected for staining microscopy,isolation and culture strain identification,and nucleic acid constant temperature amplification real-time fluorescence method and fluorescence quantitative PCR detection.The sensitivity and specificity of the two methods were obtained with culture as the gold standard,and the detection efficiency of the two methods was compared.Results Sputum samples of 322 suspected tuberculosis patients were collected.91 specimens were identified by culture and isolated Mycobacterium tuberculosis strains,with a positive rate of 28.26%;56 specimens were detected positive by microscopy,with a positive rate of 17.39%;112 specimens were detected positive by nucleic acid constant temperature amplification and real-time fluorescence,with a positive rate of 34.78%.118 cases(36.65%)were positive by fluorescence quantitative PCR.Using culture identification as the gold standard,the sensitivity and specificity of real-time fluorescence and quantitative PCR were 84.62% and 86.81%,84.85% and 83.12%,respectively.The total agreement rate between the two methods was 93.17%.Conclusion Real-time fluorescence and fluorescence quantitative PCR were used to detect Mycobacterium tuberculosis in sputum samples of suspected tuberculosis patients.Nucleic acid constant temperature amplification real-time fluorescence method is more rapid,simple and suitable for basic laboratory use.