四种重要致病性分枝杆菌DHPLC检测鉴别方法的建立  

Establishment of the denaturing high-performance liquid chromatography combined with multiplex nucleic acid amplification method for rapid identification of four important pathogenic mycobacteria

作　　者：陈茹[1] 毕英佐[2] 刘志玲[2] 刘志辉[3] 马静云[2] 曾碧健[1] 吴晓薇[1] 周科[1] 林志雄[1] 

机构地区：[1]广东出入境检验检疫局,广州510623 [2]华南农业大学 [3]广州市胸科医院

出　　处：《中国人兽共患病学报》2010年第1期41-45,52,共6页Chinese Journal of Zoonoses

基　　金：国家质检总局科技项目(2006IK019)

摘　　要：目的运用多重核酸扩增（PCR）联合变性高效液相色谱（denaturing high-performance liquid chromatography,DHPLC）分析技术原理,针对结核分枝杆菌、牛分枝杆菌、禽分枝杆菌以及副结核分枝杆菌建立多重PCR-DHPLC快速检测方法,实现同时检测鉴别四种重要致病性分枝杆菌。方法根据四种分枝杆菌特异基因序列分别设计菌种特异核酸扩增引物,通过筛选优化试验建立四重核酸扩增体系,对核酸扩增产物采用DHPLC设备进行检测分析,每一菌种的核酸扩增产物分别形成DHPLC特征峰图。对结核分枝杆菌等51株分枝杆菌标准株和分离株样品以及沙门氏菌等22株常见微生物样品进行特异性检测试验 对四种分枝杆菌特异的核酸扩增产物分别制备克隆质粒,通过对梯度稀释的阳性质粒的检测试验,进行多重PCR-DHPLC灵敏度测试 对疑似病人痰液样品和疑似发病牛的组织样品进行检测,并与细菌分离培养法进行比较以验证临床检测效果。结果该方法能快速检测鉴别上述四种分枝杆菌,检测灵敏度达到102~103基因拷贝,从131份疑似病人临床样品中检出91份结核分枝杆菌阳性,从40份来自疑似发病牛群的临床样品中检出31份牛分枝杆菌阳性,检出率均高于细菌分离培养法。结论研究表明所建立的多重PCR-DHPLC方法能快速检测鉴别四种重要致病性分枝杆菌,为人畜结核、副结核病诊断提供一种新型分子生物学技术手段。A new molecular method for simultaneously rapid detection and differentiation of Mycobacterium tuberculosis,Mycobacterium bovis,Mycobacterium avium and Mycobacterium paratuberculosis was established by using denaturing high-performance liquid chromatography（DHPLC）combined with multiplex nucleic acid amplification.These 4 important pathogenic mycobacteria were identified by separation of 4 specific PCR-amplified target fragments by DHPLC analysis.A total of 51 Mycobacterium strains and 22 other bacterial species were tested to confirm the specificity of the multiplex PCR-DHPLC assay.The sensitivity of the assay was as low as 102-103 gene copies.This method rapidly identify the positive clinical samples from human and bovine with higher detection ratio than traditional culture method and was able to identify simultaneously four pathogenic Mycobacterium,which provided a new molecular tool for rapid detection of tuberculosis and paratuberculosis in human and animals.

关 键 词：变性高效液相色谱 结核分枝杆菌 牛分枝杆菌 禽分枝杆菌 副结核分枝杆菌 多重PCR 

分 类 号：R535[医药卫生—内科学]