耐药结核分枝杆菌多重Taqman-MGB探针检测方法的建立
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摘要
目的建立耐药结核分枝杆菌的多重Taqman-MGB探针检测体系。方法针对结核分枝杆菌抗结核一线药物突变位点[利福平:Rpo B(526)/(531);乙胺丁醇:emb B(306);异烟肼:Kat G(315)、inh A(-15);链霉素:Rpsl(43)]设计并合成引物探针;构建耐药结核分枝杆菌野生型和突变型模板,对其进行梯度稀释;配制结核分枝杆菌耐药基因检测试剂,用不同类型模板进行检测;检测72例结核样本(耐利福平20株,耐异烟肼22株,耐链霉素1株,耐多药17株),并与罗氏药效试验结果比较。结果该检测体系可同时检测6个结核突变位点,最低检测限为10~4拷贝/m L,对临床样本检测其总符合率为100%,一致性好(x~2=137.3,P<0.001)。结论本研究建立了耐药结核分枝杆菌的快速检测方法,可较好应用于卫生检疫系统和临床实验室。
Objective To establish a multiple Taqman-MGB probe detection system for drug resistant Mycobacterium tuberculosis.Methods The primers and probes targeting mutation sites of Mycobacterium tuberculosis against tuberculosis first-line drug [rifampicin:Rpo B(526)/(531);ethambutol:emb B(306);isoniazid:Kat G(315),inh A(-15);streptomycin:Rps L(43) ]were designed and synthesized.Wild-type and mutant templates of drug resistant Mycobacterium tuberculosis were constructed and diluted.Drug resistance gene detection reagent of Mycobacterium tuberculosis was prepared,and tested by different templates.72 tuberculosis samples(rifampin resistance 20 strains;streptomycin resistance 22 strains;isoniazid resistance 1 strain;multiple drug resistance 17 strains) were analyzed by the reagent.The results were compared with Roche efficacy test.Results The detection system could simultaneously detect 6 tuberculosis mutation sites,and the minimum detection limit was 10~4 copies/m L.In clinical samples,the total compliance rate was 100% with good consistency(x~2= 137.3,P < 0.001).Conclusion This study establishes a rapid detection method of drug resistant Mycobacterium tuberculosis,which can be used for health quarantine system and clinical detection.
Objective To establish a multiple Taqman-MGB probe detection system for drug resistant Mycobacterium tuberculosis.Methods The primers and probes targeting mutation sites of Mycobacterium tuberculosis against tuberculosis first-line drug [rifampicin:Rpo B(526)/(531);ethambutol:emb B(306);isoniazid:Kat G(315),inh A(-15);streptomycin:Rps L(43) ]were designed and synthesized.Wild-type and mutant templates of drug resistant Mycobacterium tuberculosis were constructed and diluted.Drug resistance gene detection reagent of Mycobacterium tuberculosis was prepared,and tested by different templates.72 tuberculosis samples(rifampin resistance 20 strains;streptomycin resistance 22 strains;isoniazid resistance 1 strain;multiple drug resistance 17 strains) were analyzed by the reagent.The results were compared with Roche efficacy test.Results The detection system could simultaneously detect 6 tuberculosis mutation sites,and the minimum detection limit was 10~4 copies/m L.In clinical samples,the total compliance rate was 100% with good consistency(x~2= 137.3,P < 0.001).Conclusion This study establishes a rapid detection method of drug resistant Mycobacterium tuberculosis,which can be used for health quarantine system and clinical detection.
引文
[1]黄维忠,陈玲.耐多药结核病治疗的研究[J].中外医疗,2015,34(2):116-117.
[2]WHO.Global tuberculosis report 2013[R].Switzerland:Geneva.WHO/HTM/TB/2013.
[3]王黎霞,成诗明,陈明亭,等.2010年第五次全国结核病流行病学抽样调查报告[R].中国防痨杂志,2012,34(8):485-508.
[4]王维宁.荧光定量PCR技术检测结核杆菌临床应用分析[J].实用预防医学,2012,19(9):1404-1406.
[5]安立群.中国结核病控制工作回顾及展望[J].职业与健康,2014,30(6):842-843.
[6]Palomino JC,Martin A,Portaels F.Rapid drug resistance detection in Mycobacterium tuberculosis:a review of colourimetric methods[J].Clin Microbiol Infect,2007,13(8):754-762.
[7]孙炳奇,张娟,孙秀华.MGIT960法和绝对浓度法对耐多药结核杆菌药敏检测比对分析[J].广东医学,2014,35(12):1877-1879.
[8]李恒德,左建宏,王麓山.结核分枝杆菌耐药机制研究进展[J].现代生物医学进展,2013,13(11):2175-2176.
[9]刘志广,郭倩,魏剑浩.结核分枝杆菌gyr A基因突变与氧氟沙星耐药水平的相关性研究[J].实用预防医学,2015,22(6):641-644.
[10]刘佳庆,张丽霞,孙海柏,等.Xpert MTB/RIF在结核病诊断中的研究进展[J].广东医学,2016,37(12):1894-1899.
[11]何进才,周丹,劳诗欣.PCR反向点杂交法在结核杆菌耐药突变基因检测中的应用[J].广东医学,2013,34(3):395-397.
[12]梅早仙,韩骏锋,吴琦.结核分枝杆菌耐药性检测新进展[J].国际呼吸杂志,2015,35(8):611-615.
[2]WHO.Global tuberculosis report 2013[R].Switzerland:Geneva.WHO/HTM/TB/2013.
[3]王黎霞,成诗明,陈明亭,等.2010年第五次全国结核病流行病学抽样调查报告[R].中国防痨杂志,2012,34(8):485-508.
[4]王维宁.荧光定量PCR技术检测结核杆菌临床应用分析[J].实用预防医学,2012,19(9):1404-1406.
[5]安立群.中国结核病控制工作回顾及展望[J].职业与健康,2014,30(6):842-843.
[6]Palomino JC,Martin A,Portaels F.Rapid drug resistance detection in Mycobacterium tuberculosis:a review of colourimetric methods[J].Clin Microbiol Infect,2007,13(8):754-762.
[7]孙炳奇,张娟,孙秀华.MGIT960法和绝对浓度法对耐多药结核杆菌药敏检测比对分析[J].广东医学,2014,35(12):1877-1879.
[8]李恒德,左建宏,王麓山.结核分枝杆菌耐药机制研究进展[J].现代生物医学进展,2013,13(11):2175-2176.
[9]刘志广,郭倩,魏剑浩.结核分枝杆菌gyr A基因突变与氧氟沙星耐药水平的相关性研究[J].实用预防医学,2015,22(6):641-644.
[10]刘佳庆,张丽霞,孙海柏,等.Xpert MTB/RIF在结核病诊断中的研究进展[J].广东医学,2016,37(12):1894-1899.
[11]何进才,周丹,劳诗欣.PCR反向点杂交法在结核杆菌耐药突变基因检测中的应用[J].广东医学,2013,34(3):395-397.
[12]梅早仙,韩骏锋,吴琦.结核分枝杆菌耐药性检测新进展[J].国际呼吸杂志,2015,35(8):611-615.