耐热AKP在高GC含量细菌染色体杂交方法中的应用  

Application of Heat-resisting Alkaline Phosphatase on Quantitative DNA-DNA Hybridization among Bacteria

作　　者：黄新祥[1] 许化溪[2] 王卉放[1] 王胜军[2] 王锁英[3] 喻杰[2] 夏圣[2] 马洁[1] 许文荣[2] 江崎孝行[4] 

机构地区：[1]江苏大学医学技术学院生物化学与临床生化教研室 [2]江苏大学检验医学研究所免疫研究室,江苏镇江212001 [3]江苏大学附属医院儿科,江苏镇江212001 [4]日本岐阜大学医学部微生物学讲座

出　　处：《江苏大学学报（医学版）》2004年第416期465-469,共5页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省自然科学基金资助(BK2000029);江苏省"333工程"资助;江苏省六大人才高峰项目

摘　　要：目的应用耐热碱性磷酸酶(AKP)于微孔板DNADNA杂交技术,建立高G+C含量细菌染色体DNA同源性测定的特异性方法,为此类细菌的分类与鉴定奠定基础。方法细菌染色体DNA经分离纯化并变性后,直接非共价结合于固相微孔板中,与标有光标生物素的DNA探针进行杂交。借助抗生物素蛋白与生物素的特异性结合,通过与板上带有生物素的杂交体作用,使得链霉亲和素连接的耐热AKP固定于板上,根据底物加入后的酶促反应强度,定量判读杂交结果。结果微孔板中1μg/孔DNA的包被浓度与25～50ng/孔的生物素探针呈现较为理想的杂交结果,pH95TrisHcl缓冲液系耐热AKP的最佳作用条件。应用该法于高G+C含量的分枝杆菌种间染色体同源性分析表明,方法具有敏感、特异、简便等优点。结论耐热AKP应用于微孔板定量杂交,可不受较高杂交和测定温度的影响,适用于高G+C含量的结核杆菌等分枝杆菌属以及假单胞菌属的定性检测和同源性的定量分析。Objective: Using heat-resisting alkaline phosphatase to establish the method of microplate quantitative DNA-DNA hybridization for examining the homology among bacterial strains with high G+C percent. Methods: The denatured bacterial chromosome DNA linked to microplate reacted to the probe which was labeled by photobiotin. After adding streptoavidin labeled heat-resisting alkaline phosphatase, the enzyme was bond to microplate through combination of streptoavidin and photobiotin. The results of hybridization could be read by fluorescence analyzer following the relevant substrate was added. Results: The favorable hybridization results could be obtained when 1 μg/well covered DNA and 25～50 ng/well probe was used respectively, and pH 9.5 Tris-HCl buffer was the optimum condition for reaction of enzyme and substrate. The results showed that the method was sensitive, specific and simple, and it will adapt to determine the homology of bacteria with high G+C content. Conclusion: It had not obviously influence over high temperature of hybridization and determination when the heat-resisting alkaline phosphatase was used for quantifing DNA-DNA similarity value among bacteria, especially in Mycobacterium and Pseudomonas possessed high G+C content. [

关 键 词：耐热AKP 定量杂交 细菌 

分 类 号：R33-33[医药卫生—人体生理学]