摘要
目的以结核分枝杆菌的IS6110基因为特异性分子扩增靶标,利用TaqMan实时荧光定量PCR检测动物组织中结核分枝杆菌,以期作为科研或临床组织标本中结核分枝杆菌的检测方法。方法从155份小鼠、豚鼠和恒河猴的肺和脾组织中提取DNA,用TaqMan实时荧光定量PCR检测结核分枝杆菌IS6110基因,计算方法的灵敏度和特异度。结果实时荧光定量PCR对组织中结核分枝杆菌的最低检出限为1CFU/ml组织研磨液,其灵敏度和特异度分别为95.6%(95%CI:89.5%-98.4%)和100%(95%CI:89.6%-100%);相比而言,培养法的灵敏度和特异度分别为82.3%(95%CI:73.7%-88.6%)和100%(95%CI:89.6%-100%)。两种方法的kappa系数为0.762(95%CI:0.79-0.965)。结论TaqMan实时荧光定量PCR能快速检测感染组织中的结核分枝杆菌,具有高灵敏度和特异度,适用于科研和临床实验室对组织标本中结核分枝杆菌的检测。
Objective To use IS6110-targeted TaqMan real-time fluorescent quantitative PCR(TaqMan real-time FQPCR)to quantitatively detect Mycobacterium tuberculosis from animal tissues.Methods DNA was extracted from a total of 155 spleen and lung tissue specimens from animals(mice,guinea pigs,and Rhesus monkeys).TaqMan real-time FQ-PCR was used to detect the IS6110 gene of M.tuberculosis.The sensitivity and specificity of the method were evaluated.Results The detection limit of the TaqMan real-time FQ-PCR was 1CFU per ml of tissue homogenate solution.This method had a high level of sensitivity of 95.58%(95%CI:89.47%-98.36%)and a high level of and specificity of100%(95%CI:89.56%-100%),respectively.In comparison,culturing M.tuberculosis has a sensitivity of 82.3%(95%CI:73.7%-88.6%)and a specificity of 100%(95%CI:89.6%-100%).The kappa coefficient of the two methods was 0.762(95%CI:0.79-0.965).Conclusion The TaqMan real-time FQ-PCR is a method for rapid detection of M.tuberculosis in infected tissues.This technique has a high level of sensitivity and specificity.Thus,this technique is suitable for use in research and clinical laboratories.
作者
孟凤珍
郭铭
周立
高建峰
霍文哲
MENG Feng-zhen;GUO Ming;ZHOU Li;GAO Jian-feng;HUO Wen-zhe(Wuhan University Schoolof Basic Medical Sciences,Center for Animal Experimentation/Animal Biosa fety Level 3 Laboratory,Wuhan 430071,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第9期769-774,共6页
Journal of Pathogen Biology
基金
国家十二五艾滋病和病毒性肝炎等重大传染病防治科技重大专项(No.2012ZX10004501-001-004,2013ZX10003009-003)
国家自然科学基金项目(No.81571962)
国家自然科学基金青年项目(No.81301428)
湖北省自然科学基金项目(No.2014CFA076)
中国博士后科学基金项目(No.2015M572199)
武汉大学自主科研项目(No.2042015kf0188)
