毛细管内均相辣根过氧化物酶(HRP)与核酸杂交偶联HRP催化化学发光比较研究  

Differences between coupling of nucleic acid hybridization and catalytic chemiluminescence of free horseradish peroxidase in capillary tube

作　　者：向安[1] 汪钦[1] 包晗[1] 颜真[1] 郭晏海[1] 

机构地区：[1]第四军医大学全军基因诊断技术研究所,陕西西安710032

出　　处：《现代生物医学进展》2011年第5期857-860,共4页Progress in Modern Biomedicine

基　　金：国家"十一五""艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2009ZX10004-311);陕西省自然科学基金项目(2009JM4005)

摘　　要：目的:了解毛细管内核酸偶联和游离辣根过氧化物酶(HRP)催化化学发光差异。方法:不同浓度HRP和经核酸杂交固定于毛细管内壁HRP催化化学发光反应。结果:①游离HRP催化化学发光线性检测范围窄(2.7×10-5-1.3×10-6mg/ml,R2>0.96),下限为1.0×10-7mg/ml;②2.0×1014-2.0×106copies/ml的5μl单链DNA杂交后,1.0-10min时DNA浓度M对数(lgM)与化学发光I值线性相关(R2>0.99),且大于阴性I值平均数+3倍标准偏差(s.d);③5.0×1011-5.0×106copies/ml的5μl PCR产物杂交后,10min内PCR产物对数lgM与I值线性相关(R2>0.97),且大于阴性I值平均数+3倍标准偏差(s.d)。4.0-7.0min内lgM与I值的R2>0.99,3次平行检测标准偏差<5.0%。结论:毛细管内核酸杂交的HRP催化化学发光检测线性范围宽、灵敏度高、底物用量少,有望用于临床核酸分子杂交检测。Objective：To investigate the characteristic of chemoluminescence under the catalytic effects of Horseradish peroxidase（HRP） in capillary tube.Methods：Different concentration of HRP that free or conjugated to capillary tube by nucleic hybridization been used to catalyze chemiluminescence reaction.Results：① The linear range of the free HRP was limited（2.7×10-5-1.3×10-6mg/ml,R20.96）,and the absolute detection limit was 1.0×10-7mg/ml.②The chemiluminescence detected value（I） was higher than the sum of means of there background observations and triple standard deviations（s.d）,and had a positive linear correlation with the level of logarithmic DNA concentration（R20.99）,when DNA sequences from2.0×1014 to 2.0×106copies/ml.③ From 5.0×1011 to 5.0×106copies/ml,the logarithmic value of concentration（lgM） of 5ul products of PCR was higher than the sum of means of there background observations and triple standard deviations（s.d）,and had a positive linear correlation with the value（I）,R20.97.In addition,within 4.0-7.0min,R20.99,and the relative standard deviation is less than 5.0%.Conclusion：This study suggest that the chemiluminescence detection of nucleic hybridization in capillary tube with catalyze of HRP is a rapid,simple,and micro-nucleic detection technology with sensitivity and specificity,hence,might be clinically used in the future.

关 键 词：微体系 固定化酶 辣根过氧化酶 化学发光 

分 类 号：O657.8[理学—分析化学] Q814[理学—化学]