多重PCR-变性高效液相色谱法快速检测结核致病菌群并同步鉴别致病菌种

Simultaneous Detection of Mycobacterium tuberculosis Complex and Identification of Mycobacterium Species by Multiplex PCR Combined with Denaturing High-performance Liquid Chromatography

本研究旨在运用多重PCR联合变性高效液相色谱(DHPLC)技术原理,建立快速检测人兽结核致病菌群并鉴别主要致病菌种的新方法。建立了四重PCR-DHPLC方法,可同时检测结核分枝杆菌复合群(MTC)特异的IS6110和IS1081插入序列、结核分枝杆菌特异结构基因和牛分枝杆菌特异结构基因共4个目标基因。采用13种分枝杆菌标准菌株和分离株以及23种常见微生物样品进行特异性试验,采用纯化标准菌株DNA以及克隆了扩增序列的重组质粒DNA进行检测灵敏度试验,采用从临床疑似发病牛群采集的牛组织样品进行临床检测试验,并与细菌分离培养方法进行比对。结果表明:所建立的多重PCR-DHPLC方法能特异检测MTC并准确鉴别结核分枝杆菌和牛分枝杆菌,而对其它11种分枝杆菌以及23种常见微生物样品均呈典型阴性检测结果;检测灵敏度达到每个反应102～103基因拷贝或3.6～6.8fg DNA模板浓度。采用该法从39份临床疑似样品中检出33份MTC阳性并检出其中28份为牛分枝杆菌阳性,而培养法检出21份阳性样品。采用该法临床检测全程可缩短至1d之内,其中对纯化DNA样品的检测分析可在2h内完成。本研究为人兽结核致病菌(群)的快速检测鉴别提供了新型分子生物学方法。

Based on multiplex PCR combined with denaturing high-performance liquid chromatog- raphy technique, this study was carried out for simultaneous detection of Mycobacterium tubercu- losis complex and identification of major pathogenic species for human and animal tuberculosis. A novel multiplex PCR-DHPLC method was established which identified four gene targets in an re- action,including IS6110 and IS1081 insertion sequence specific for Mycobacterium tuberculosis complex and specific structure gene for M. tuberculosis and M. boris respectively. The specifica- tion of the quadruplex PCR-DHPLC assay was verified by tests on 13 species of mycobacteria ref- erence and isolated strains and 23 species of other microorganism. The limit of detection wasmeasured by testing on purified mycobacterium DNA and cloned plasmid DNA. Clinical perform- ance of the assay was demonstrated by detection on bovine tissue samples from infected herds and compared with culture. Results were as follows. The assay specifically detected strains of Myco- bacterium tuberculosis complex and simultaneously differentiated M. tuberculosis and M. boris strains. The limit of detection on cloned plasmid DNA was 102 to 103 gene copy or 3.6 to 6.8 fg DNA per reaction. For the detection on 39 suspected tissue samples, the assay detected 33 sam- ples positive for Mycobacterium tuberculosis complex and identified 28 as M. boris positive, while 21 samples were showed positive by culture method. The whole detection procedure of the multi- plex PCR-DHPLC assay on clinical samples could finish within 1 day, and the detection on puri- fied DNA sample took around 2 hours. This study provided a novel rapid molecular method for differential identification of pathogenic mycobacteria for human and animal tuberculosis.