可视化膜芯片技术用于结核分枝杆菌基因分型的研究

Using membrane chip to genotype Mycobacterium tuberculosis by spoligotyping

目的建立用于结核分枝杆菌间隔区寡核苷酸分型的膜芯片技术,并评估其应用价值。方法根据结核分枝杆菌直接重复序列43间隔子设计特异的分型探针并制作膜芯片,通过直接重复序列特异引物扩增基因组间隔子相应区段的DNA。扩增引物用生物素标记,扩增标记后的产物与芯片的探针杂交,根据显色后产生的斑点确定标本的基因亚型。对175株临床分离菌株用该方法进行分型,用传统膜杂交法作对照。结果H37Rv和BCG菌株DNA扩增产物与膜芯片杂交,43条探针均出现杂交斑点。用膜芯片技术杂交获得H37Rv和BCG菌株的基因亚型,与传统固相膜杂交结果一致。用该法对175株临床分离菌株进行分型,其中167株属于4个已知的基因家族,最大的一个为北京家族,占62.9%(110/175)。北京家族中有107株为典型北京家族(1-34间隔区缺失),3株为北京样基因型(Beijing-like),即非典型北京家族(35-43间隔区也有缺失)。T家族占30.9%(54/175),包括34株T1,14株T2,6株T3。2株Manu2和1株LAM9;未匹配8株,占4.6%(8/175)。膜芯片技术杂交结果与传统膜杂交结果完全相同,耗时由膜杂交法的195 min缩短为22 min。方法的灵敏度为10个MTB/ml。结论膜芯片技术用于结核分枝杆菌间隔区寡核苷酸基因分型灵敏度高且简便易行,适用于临床流行病学追踪与分析。

Objective To establish a membrane chip strategy to genotype Mycobacterium tuberculosis by spoligotyping and evaluate its clinical application.Methods The membrane chip was made by designing specific hybriding probes based on the 43 spacers of the direct repeat sequence of Mycobacterium tuberculosis.Sequences of the genomic spacer were amplified by direct repeat specific primers that labeled with biotin.The amplified products were hybridized with the probes on the chip.The genotypes of the sample were determined according to the dots produced by color reaction.Total 175 clinical isolates were typed and compared with traditional hybridization method.Results The PCR products of H37Rv and BCG were hybridized with membrane and the spots were found in 43 probes.The subtypes of H37Rv and BCG obtained by membrane chip technique were as same as traditional membrane hybridization method.175 clinical isolates were typed by membrane chip technique,of which 167 belonged to 4 known gene families.The largest group was Beijing family,accounting for 62.9%(110/175).There are 107 typical Beijing families(1-34 spacer deletion)and 3 Beijing-like genotypes(besides spacer 1-34,deletion also found in 35-43).T family accounted for 30.9%(54/175),including 34 strains of T1,14 strains of T2 and 6 strains of T3.There were two strains of Manu 2 and one strain of LAM9.The remaining 8 strains did not match in the database,accounting for 4.6%(8/175).The hybridization results of membrane chip technique were identical with those of traditional membrane hybridization,and the time consumed was shortened from 195 min to 22 min.The sensitivity of the recommended method is 10 MTB/ml.Conclusion Membrane chip technique is highly sensitive,simple and feasible for spoligotyping of Mycobacterium tuberculosis,and is suitable for clinical epidemiological tracing and analysis.