建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

Development of a liquidchip assay for simultaneous detection of Mycobacterium tuberculosis complex and M. avium and M. paratuberculosis

摘要:

运用液相芯片技术原理, 以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因, 设计筛选4套扩增引物和杂交探针, 建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示, 四重液相芯片方法可特异检测鉴别目标菌种(群), 与其它分枝杆菌菌种或微生物无非特异交叉反应; 检测敏感性达2.1×101?2.5×102基因拷贝或0.06?0.74 fg DNA; 组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌, 检出率分别达75.6% (99/131)和94.9% (37/39), 显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示, 四重液相芯片方法与荧光PCR方法的阳性符合率为83% (24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d, 其中对纯化DNA模板的检测时间可在2?3 h内完成。

Abstract:

A microsphere-based LiquidChip assay was developed for rapid and simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium avium subsp. paratuberculosis (MAP). Four sets of oligonucleotide primers and probes which respectively targeted at IS6110, IS1081, IS1245 and F57 specific genes of mycobacterium were selected to establish the quadruplex assay. The quadruplex assay specifically identified target strains from a total of 54 strains of 13 species of mycobacterium, and 23 species of non-mycobacterium microorganisms were all detected as negative. The sensitivity on detecting cloned plasmid DNA by the quadruplex assay was 2.1×101?2.5×102 genomic copies or 0.06?0.74 fg DNA per reaction. The intra-assay and inter-assay variations of the quadruplex assays were both lower than 10%. The assay detected 75.6% (99/131) and 94.9% (37/39) positive from TB suspected human sputum samples and bovine tissue samples respectively, compared to culture methods that detected 38.9% and 53.8% positive from human and bovine samples respectively. The quadruplex assays also detected 24 MAP positive from 29 bovine blood specimens detected as positive by real time PCR specific for MAP. The tests on quadruplex mixed templates showed that the assay could identify mix infections. Clinical detection by the assay could be finished within one day, and the detection on purified DNA template could be completed in 2?3 h.