液相芯片技术联合等位基因特异性引物延伸反应检测结核分枝杆菌耐多药突变  

Detection of multidrug-resistant Mycobacterium Tuberculosis by suspension array technology and allele-specific primer extension reaction

作　　者：李婧婵[1] 姜春来[2] 于源华[1] 宋海鹏[1] 

机构地区：[1]长春理工大学生命科学技术学院,长春130022 [2]吉林大学生命科学学院,长春130012

出　　处：《临床检验杂志》2016年第3期165-168,共4页Chinese Journal of Clinical Laboratory Science

基　　金：吉林省科技发展计划项目基金(20130102086JC)

摘　　要：目的建立一种耐多药结核分枝杆菌检测方法。方法设计并构建含有kat G315、rpo B526、rpo B531耐药基因突变位点的质粒;通过PCR扩增及纯化、等位基因特异性引物延伸反应（ASPE）、荧光微球杂交反应及液相芯片系统Luminex 200检测;并对该方法反应条件进行探索及方法学评价。结果 ASPE反应中Tsp DNA聚合酶最适浓度为0.025 U/μL,杂交荧光微球浓度为每μL 100个,链霉亲和素藻红蛋白浓度及杂交时间分别为2μg/m L和20 min;所建立方法的检测灵敏度最低可达1ng/m L;当质粒浓度为1.5×105ng/m L时,批间和批内变异系数（CV）分别为6.48%～12.15%和0.35%～6.92%;当质粒浓度为2 ng/m L时,批间和批内变异系数（CV）分别为5.73%～10.77%和0.97%～8.91%;特异性引物探针与其他突变位点均无交叉反应。结论液相芯片技术联合等位基因特异性引物延伸反应检测耐多药结核分枝杆菌灵敏且具有成本低、高通量等特点,极具临床应用潜力。Objective To establish a method for the detection of multidrug-resistant Mycobacterium Tuberculosis. Methods The plasmids carrying the drug-resistant mutation genes,including kat G315,rpo B526 and rpo B531 gene locus,were designed and constructed.Then,the samples containing the plasmids were detected by PCR amplification and purification,allele-specific primer extension（ ASPE） reaction,fluorescence microsphere hybridization and suspension array Luminex 200 system step-by-step. The reaction conditions of the established method were further optimized,and its methodological evaluation was performed. Results The optimal concentration of Tsp DNA polymerase in ASPE reaction was 0. 025 U / μL,the concentration of fluorescence microspheres in the hybridization was 100 / μL,and the concentration of Streptavidin-R-Phycoerythrin and hybridization time were 2 μg / m L and 20 min,respectively.The minimum detectable concentration of the established method was 1 ng / m L. When the concentration of plasmids in samples was1. 5 × 105 ng / m L,the inter-batch and intra-batch coefficients of variation（ CVs） were 6. 48%-12. 15% and 0. 35%-6. 92%,respectively. When the concentration of plasmids in samples was 2 ng / m L,the inter-batch and intra-batch CVs were 5. 73%-10. 77% and0. 97%-8. 91%,respectively. There was no cross reaction between the specific primer probe and other mutant sites. Conclusion The established method for the detection of multidrug-resistant Mycobacterium Tuberculosis based on suspension array technology and ASPE reaction is characterized as high sensitivity,low cost and high-throughput,which may be used to diagnose the infection of multidrug-resistant Mycobacterium Tuberculosis.

关 键 词：液相芯片技术 等位基因特异性引物延伸反应 结核分枝杆菌 耐多药性 

分 类 号：R446.5[医药卫生—诊断学]