应用引物原位标记技术快速检测SOX2基因

Rapid detection of SOX2 gene by primed in situ labeling

目的应用引物原位标记（primedin situ labeling，PRINS）代替荧光原位杂交技术对SOX2基因进行快速检测。方法常规制备人外周血染色体并制片，利用SOX2基因特异性引物在染色体上原位扩增包含生物素标记的探针。用酪胺信号放大（tyramidesignalamplification，TSA）生物素系统处理经标记的探针。最后用链霉亲和素-德克萨斯红（streptavidin-Texasred）对生物素信号进行荧光染色，用DAPI染料对染色体进行复染。作为对照，MCF-10F细胞用慢病毒载体转染导入SOX2基因，使用相同的方法检测结合位点。结果VideoTesT—FISH软件分析提示，实验组3号染色体长臂端有特异红色荧光信号表达，空白组与未经TSA信号处理的对照组则无特异性的红色荧光信号。经过转染的MCF-10F细胞则表现出不同的SOX2基因整合效果。结论PRINS技术利用高敏感度的原位PCR技术（insituPCR）结合荧光标记的脱氧核苷酸可在细胞内原位合成形成探针，可大大减少探针的费用与检测的时间，提高检测效率。在诱导多能干细胞的鉴定与分类中具有较为广泛的应用前景。

Objective To rapidly detect SOX2 gene using primed in situ labeling （PRINS）. Methods Human peripheral blood samples were cultured using an optimized method. Sequence of the SOX2 gene was amplified in situ with biotin-labeled specific primers and processed with a tyramide signal amplification （TSA） biotin system. Subsequently, fluorescence-stained signal was detected by streptavidin-Texas red. For the control group, MCF-10F cells were transfected with Lentivirus hSox2. Results By VideoTesT- FISH software analysis, the long arm of chromosome 3 in the experimental group showed a specific red fluorescence signal, whilst the control samples showed no specific signals for SOX2. Transfected MCF-10F cells showed various efficiency of SOX2 gene integration. Conclusion PRINS utilizes a highly sensitive in situ PCR technique combined with fluorescence labeled oligodeoxynucleotides can synthesize probes in situ, thus greatly reducing the cost of probe and time for detection. It can facilitate identification and classification of induced pluripotent stem cells, and has many potential applications in this prospect.