基于核酸外切酶Ⅲ降解原理荧光检测乙肝病毒HBV  

Fluorescence Detection of Hepatitis B Virus Amount Coupled with Exonuclease Ⅲ Cleavage

作　　者：牛淑妍[1] 渠丽景[1] 

机构地区：[1]青岛科技大学化学与分子工程学院,山东青岛266042

出　　处：《青岛科技大学学报（自然科学版）》2010年第6期579-582,共4页Journal of Qingdao University of Science and Technology:Natural Science Edition

基　　金：青岛科技大学博士启动基金项目(0022149)

摘　　要：利用核酸外切酶Ⅲ降解杂交的DNA链,将荧光素修饰的DNA探针释放到溶液中,通过测定溶液的荧光强度来测定乙肝病毒(HBV)的浓度。考察了缓冲溶液的种类、pH值、反应时间等对反应体系荧光强度的影响,确定了最佳反应条件:最佳缓冲溶液为TT缓冲溶液(pH8.0,250 mmol.L-1Tris-HCl,体积分数0.1%Tween 20),缓冲溶液的最佳pH值为9.0,生物素与亲和素最佳结合时间为25 min,DNA的最佳杂交时间为20 min,酶反应最佳时间为1.1 h。在最佳反应条件下,随着HBV浓度的增加,体系的荧光强度明显增强。该方法测定HBV的线性范围为0.5～5.0 pmol.L-1,检测限为0.335 pmol.L-1。The digestion and degradation of the dsDNA to a single chain by exonuclease Ⅲ were determined by fluorescence spectroscopy.HBV can be detected by measuring the fluorescence intensity of the solution analyzed.The best reaction conditions were obtained through the study of the effect of different buffer solutions,different pH and different reaction time under the system＇s fluorescent intensity.TT buffer（pH 8.0,250 mmol·L^-1 Tris-HCl,volume fraction of Tween 20 0.1%）was chosen as the reaction buffer,the pH of TT buffer was 9.0,the reaction time of biotin and streptavidin was 25 min,20 min was chosen as the hybridization time of capture probes and target DNA,1.1 h was the enzyme incubation time.The effect of HBV concentration on the system＇s fluorescent intensity under the best reaction conditions was also studied.The fluorescence intensity of the system increased with the concentration of HBV,whose concentration of HBV can be detected.In the system of fluorescence spectroscopy based on exonuclease Ⅲ,the HBV could be quantified in the ranges of 0.5—5.0 pmol·L-1 and the detection limit is 0.335 pmol·L-1.

关 键 词：HBV 核酸外切酶Ⅲ 荧光光谱 

分 类 号：O657.32[理学—分析化学]