芯片显色法检测结核分枝杆菌利福平和异烟肼耐药基因实验条件优化研究  

Optimization of experimental condition to detect the resistance genes of rifampin(RFP) and isoniazid (INH) in Mycobacterium tuberculosis by using Coloration gene chips

作　　者：王万相 郭乃洲 严爱华 马达 马力 周步全 景奉香[3] 唐向荣[3] 朱殊 

机构地区：[1]盐城市第一人民医院,224006 [2]盐城市第二人民医院,224002 [3]中国科学院上海微系统与信息技术研究所,200050 [4]南通大学公共卫生学院,江苏226001

出　　处：《中国实用医药》2007年第35期13-15,共3页China Practical Medicine

基　　金：盐城市科技局;卫生局科技发展计划项目(编号YK2005105)

摘　　要：目的优化研究芯片显色法检测结核分枝杆菌利福平和异烟肼耐药基因的最佳条件。方法自制DNA芯片,每条寡核苷酸探针片段长度为19bp,点样浓度在15μmol/L,以痰液为待检标本,分别用不同引物、Mg2+、核苷酸、DNA聚合酶浓度等进行多重聚合酶链反应;分别在不同时间、温度下进行杂交,分别在不同酶作用时间和显色时间进行分析,比较不同条件芯片检测结果。结果引物、Mg2+、核苷酸、DNA聚合酶浓度分别为0.2μmol/L、1mmol/L、0.2mmol/L、5U效果最好,杂交时间和杂交温度分别为0.5h、60℃信号最强,酶作用时间和显色时间均为0.5h时得到最佳结果。结论在最佳条件下,该技术的重复性、特异性、灵敏度与可靠性均较好,适合基层医院推广应用。Objective To examine of optimum condition to detect the resistance genes of rifampin(RFP) and isoniazid(INH) in Mycobacterium tuberculosis by using coloration gene chips.Methods The DNA chip was fabricated,on which oligonucleotide bougies′length was 19 base pairs,and the concentration was above 15 μmog/L.The sputum sample were used for detection.A primer,Mg2+,ribonucleotide and DNA polymerase of different consistency were designed to compare for multiple-polymerase chain reaction.Different hybridizing time and temperature were designed to compare the hybridizing dynamics.A enzyme labeled compound of different acting time and chromogenic time were designed to compare.Results The optimum consistency of primer,Mg2+,ribonucleotide and DNA polymerase respectively were 0.2 μmol/L、1 mmol/L、0.2 mmol/L、5 U to hybridize at 60℃ for 18 h was the best condition.The enzyme labeled compound of acting time and chromogenic time at 0.5 h was the best condition.Conclusion The reproducibility,specificity,sensitivity and reliability was good in the optimum condition of experiment.It was fitting for local hospital to use.

关 键 词：痰标本 结核分枝杆菌DNA 显色法 耐药基因 基因芯片 实验条件 

分 类 号：R450[医药卫生—治疗学]