摘要目的 建立ROS1基因的非重复序列荧光原位杂交(FISH)探针,比较其与商品化普通探针在非小细胞肺癌检测方面的差异.方法 探针制备的过程中加入Human Cot-1 DNA,与基因组中的重复序列复性结合成双链,用双链特异性酶特异性的切除重复序列.用红色和绿色荧光素分别标记BAC克隆片段化的PCR产物,最终混合得到ROS1基因不含重复序列的双色探针.将制备的非重复序列FISH探针用于2009至2013年收集的经基因测序检测确定的53例ROS1基因重排的非小细胞肺癌标本,并与传统探针测定结果比较.结果 与商品化的重复序列探针比较,非重复序列FISH探针背景清晰,仅在目的区域存在明亮的荧光杂交信号,噪音明显低于商品化探针.两种探针间的杂交率差异有统计学意义(P＜0.05);两组探针之间的准确率差异无统计学意义(P＞0.05).结论 经比较显示,非重复序列的ROS1双色FISH探针在对非小细胞肺癌样本检测中明显改善探针的杂交效率和信噪比,有利于提高检测的准确度.

abstractsObjective To establish a repeat-free ROS1 gene fluorescence in situ hybridization (FISH) probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequence.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared with the commercialized probe,repeat-free FISH probe exhibited excellent efficiency and low signal to noise ratio (SNR) in samples.There was statistical significance in the difference between the hybridization rate of these two probes(P＜0.05),but there was no difference between the accuracy rate (P＞0.05).Conclusion The repeat-free ROS1 FISH probe significantly improves the probe hybridization efficiency and SNR in nonsmall cell lung cancer (NSCLC),resulting in an increased accuracy of detection.