| 向安汪钦包晗颜真郭晏海.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[J].,2011,11(5):857-860 |
| 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 catalyticchemiluminescence of free horseradish peroxidase in capillary tube |
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| DOI： |
| 中文关键词: 微体系 固定化酶 辣根过氧化酶 化学发光 |
| 英文关键词: mini-system immobilized enzyme Horseradish peroxidase chemiluminesence |
| 基金项目: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). |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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, R2>0.96), 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 (R2>0.99), 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), R2>0.97. In addition,
within 4.0-7.0min, R2>0.99, 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. |
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