核酸薄膜技术快速检测结核分枝杆菌katG突变的研究  

Rapid Detection of katG and its Mutation in Mycobacterium Tuberculosis Clinical Isolates by using Oligonucleotide Arrays

作　　者：宗兆婧[1] 刘梅[1] 陈杨[1] 王建华[1] 李娜娜[1] 张建勇[1] 张泓[1] 陈玲[1] 

机构地区：[1]遵义医学院附属医院呼吸内科呼吸医学研究室,贵州遵义563003

出　　处：《中国实验诊断学》2012年第10期1809-1812,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金项目(项目批准号81160003)

摘　　要：目的研制一种新的核苷酸薄膜,通过导流杂交技术检测结核分枝杆菌katG基因突变类型,以快速判断结核分枝杆菌对异烟肼的耐受性,寻找一种快速简便检测耐异烟肼结核病的方法。方法根据结核分枝杆菌katG基因序列设计1个野生型及2个常见突变型寡核苷酸特异探针,探针5’末端连接亚甲基(-CH2)。同时设计1个人类基因组探针以及1个生物素探针。制作低密度核苷酸薄膜微阵距列,通过导流杂交技术进行杂交,将杂交结果与DNA测序法对照。结果核酸测序显示,突变位点分别出现在包括315位点在内的7个位点,315位点突变占75.0%(18/24),最常见突变形式为AGC315ACC所致Ser315Thr氨基酸改变。杂交结果与测序结果符合率为93.9%(31/33),与DNA测序相比,其阳性预测值、阴性预测值、敏感度、特异度分别为90.0%、100%、100%、86.7%。结论核酸薄膜导流杂交技术能快速、简便、高效地检测临床标本中有无结核分枝杆菌,并可提示结核分枝杆菌有无异烟肼耐药性,指导临床用药。Objective To develop a new Oligonucleotide Arrays rapidly detect the katG gene mutation in isoniazid-resistant Mycobacterium tuberculosis clinical isolate through flow-through hybridization technique.Methods One wild-type and two mutant oligonucleotide probes were designed based on Mycobacterium tuberculosis katG gene sequences,and a human genome（β-globin） probe and a biotinylated probe（Biotin） were designed as control.All of these probes have-CH2 in 5′.katG gene was amplified by biotin-labeled primers,and then hybridized with the low-density Oligonucleotide Arrays by flow-through hybridization technique.Results were compared with DNA sequencing.Results 24/86 isolates carried mutations at seven codons on the amplified fragment of the katG gene.The consistency rate of Hybridization and DNA sequencing is 93.9%.Positive predictive value,negative predictive value,sensitivity,specificity of the Hybridization is 90.0%、100%、100%、86.7%,respectively.Conclusion The Macroarray flow-through hybridization technique is a highly sensitive method for rapid detection Mycobacterium tuberculosis and mutations in katG gene,and in high line with DNA sequencing.It may be a new method to detect multi-drug resistance tuberculosis in clinical isolates.

关 键 词：结核分枝杆菌 KATG基因 异烟肼 核酸薄膜 导流杂交 

分 类 号：R450[医药卫生—治疗学]