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目的:建立应用于遗传性白内障基因突变筛查的DNA过氧化物酶检测方法。方法:针对已报道的遗传性白内障的基因突变位点晶状体蛋白CRYAB的p.R11H突变[CRYAB-32(G>A)]和缝隙连接蛋白50(Cx50)的p.S276F突变[CX50-827(C>T)],设计DNA过氧化物酶分子探针,建立DNA过氧化物酶探针和靶序列核苷酸反应体系。两个突变位点的检测均分为6组,即阳性对照组、阴性对照组、阳性实验组、阴性实验组以及2个空白对照组。主要观测各组与DNA过氧化物酶探针的显色反应。结果:DNA过氧化物酶探针与含CRYAB-32(G>A)及CX50-827(C>T)的阳性标本,在特定反应中结合均显现浅绿色,而与不含该突变的野生型DNA片段不反应不显色。结论:有过氧化物酶活性的DNA过氧化物酶作为一类新的颜色反应标签可应用于遗传性白内障基因突变的检测,能实现检测的可视化,简化检测硬件和操作步骤。
OBJECTIVE: To establish a DNA peroxidase assay for the screening of inherited cataract gene mutations. Methods: The p.R11H mutation [CRYAB-32 (G> A)] and the gap junction protein 50 (Cx50) p.S276F mutation [CX50-827 (Cx50)] of the CRYAB gene have been reported for inherited cataract C> T)], the design of DNA peroxidase molecular probe, the establishment of DNA peroxidase probe and target nucleotide reaction system. The detection of two mutation sites were divided into six groups, namely positive control group, negative control group, positive experimental group, negative experimental group and two blank control groups. The main observation of each group and DNA peroxidase probe color reaction. RESULTS: DNA peroxidase probes were positive for both CRYAB-32 (G> A) and CX50-827 (C> T) Type DNA fragment does not respond to no color. CONCLUSIONS: Peroxidase-catalyzed DNA peroxidase, as a new class of color reaction tags, can be used in the detection of inherited cataract gene mutations, enabling visualization of assays and simplifying the detection of hardware and procedures.