应用基因阵列法快速检测结核分枝杆菌rpoB基因突变

目的　研制一种新型的基因阵列 ,用于结核分枝杆菌耐利福平分离株rpoB基因突变的快速检测。方法　根据结核分枝杆菌rpoB基因序列设计寡核苷酸探针并制作基因阵列 ,用生物素标记的引物扩增结核分枝杆菌rpoB基因突变热点的目的片断 ,与基因阵列杂交 ,同时以聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序法为对照。结果　 111株结核分枝杆菌临床分离株经PCR SSCP分析 ,4 1株RFP敏感株SSCP图谱与结核分枝杆菌标准株相同 ;70株耐RFP菌株中 ,6 3株(90 % )SSCP图谱与结核分枝杆菌标准株不同 ,其余 7株SSCP图谱与结核分枝杆菌标准株相同。基因阵列检测结果 4 1株RFP敏感株杂交图谱与标准株完全相同 ,70株耐RFP临床分离株中 ,6 3株检测到rpoB基因突变 ,检出率为 90 % ;其中 37株 (5 3% ) 5 31位丝氨酸 (Ser)置换 ,15株 (2 1% ) 5 2 6位组氨酸(His)置换 ,11株 (16 % )其他位置的氨基酸置换。基因阵列检测结果与PCR SSCP及测序结果一致。结论　用基因阵列法可简便、快速、准确地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变。

Rapid detection of rpoB mutations in Mycobacterium tuberculosis by gene array

Objective To develop a new method,gene array,which can be used for rapid detection of rpoB mutations in Mycobacterium tuberculosis . Methods Probes were designed according to the sequence of Mycobacterium tuberculosis rpoB gene and the gene array was developed. The DNA fragment which contained hot mutation sites of rpoB gene was amplified with biotin-labelled primers by PCR,and then hybridized with gene array. Mycobacterium tuberculosis strain H_ 37 Rv DNA was used as the control. The rpoB genes in Mycobacterium tuberculosis clinical isolates were also analyzed by polymerase chain reaction-single stranded conformational polymorphism(PCR-SSCP) and PCR-DNA sequencing. Results We analyzed the rpoB genes of 111 Mycobacterium tuberculosis clinical isolates by PCR-SSCP. Of 70 rifampicin-resistant Mycobacterium tuberculosis isolates,63 isolates had different SSCP profiles from that of the standard strain H_ 37 Rv. No difference from the standard strain was found in 41 rifampicin-susceptible and 7 rifampicin-resistant isolates. We also analyzed their rpoB genes by gene array. Of 111 Mycobacterium tuberculosis clinical isolates,the results of gene array in 41 drug-sensitive strains were similar to that in Mycobacterium tuberculosis H_ 37 Rv. 90%(63/70) rifampicin-resistant strains had rpoB gene mutation. 53%(37/70) rifampicin-resistant strains had serine substitution at codon 531. 21%(15/70) strains had histidine substitution at codon 526. 16%(11/70) strains had amino acids substitution in other position. The results of gene array corresponded with that of PCR-SSCP and DNA sequencing. Conclusion Gene array might become a rapid,simple,and accurate method for detecting rpoB mutations in most of the rifampicin-resistant Mycobacterium tuberculosis .