CAJ | 학술논문

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10~2～10~3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.
목적 운용다중핵산확증(PCR)연합변성고효액상색보(denaturing high-performance liquid chromatography, DHPLC)분석기술원리,침대결핵분지간균、우분지간균、금분지간균이급부결핵분지간균건립다중PCR-DHPLC쾌속검측방법,실현동시검측감별사충중요치병성분지간균.방법 근거사충분지간균특이기인서렬분별설계균충특이핵산확증인물,통과사선우화시험건립사중핵산확증체계,대핵산확증산물채용DHPLC설비진행검측분석,매일균충적핵산확증산물분별형성DHPLC특정봉도.대결핵분지간균등51주분지간균표준주화분리주양품이급사문씨균등22주상견미생물양품진행특이성검측시험;대사충분지간균특이적핵산확증산물분별제비극륭질립,통과대제도희석적양성질립적검측시험,진행다중PCR-DHPLC령민도측시;대의사병인담액양품화의사발병우적조직양품진행검측,병여세균분리배양법진행비교이험증림상검측효과. 결과해방법능쾌속검측감별상술사충분지간균,검측령민도체도10~2～10~3기인고패,종131빈의사병인림상양품중검출91빈결핵분지간균양성,종40빈래자의사발병우군적림상양품중검출31빈우분지간균양성,검출솔균고우세균분리배양법.결론 연구표명소건립적다중PCR-DHPLC방법능쾌속검측감별사충중요치병성분지간균,위인축결핵、부결핵병진단제공일충신형분자생물학기술수단.
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 10~2-10~3 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.