目的 探讨在不同操作平台、染料间高分辨率熔解曲线分析(HRM)技术检测粪便基因突变的性能差异.方法 收集合格的粪便125份,其中大肠癌40份,晚期腺瘤35份,正常对照50份,在LightCycler 480与Rotor-Gene 6000平台设备上,分别使用Resolight或Eva Green染料,检测K-ras基因突变率的差异,所有样品均行测序.结果 测序发现,大肠癌组、晚期腺瘤组、正常对照组K-ras基因突变率分别是40%、31.4%、0%.在LightCycler 480平台,用Resolight/Eva Green染料突变检出率分别为40%/45%、31.4%/34.2%、0%/0%.在Rotor-Gene 6000平台,用Resolight/Eva Green染料突变检出率分别为42.5%/47.5%、34.2%/37.1%、0%/0%.在各组中,不同操作平台、染料间突变检出率差异无统计学意义(P>0.05).HRM检测粪便基因突变敏感度达100%.结论 HRM在不同操作平台、染料间的突变检出率差异无显著性.%Objective To evaluate the performance of high- resolution melting curve analysis ( HRM ) in detection of fecal mutations on different operating platforms and dyes. Methods A total of 125 qualified stool samples were collected from different subjects, including patients with colorectal cancer ( n = 40 ) and advanced adenoma ( n = 35 ), and normal controls ( n = 50 ). On both Light - Cycler 480 and Rotor - Gene 6000 platforms, Resolight or Eva Green dye was used in detection of K - ras mutation. DNA sequencing was applied as gold standard. Results DNA sequencing showed that K - ras gene mutation rates were 40, 31. 4 and 0% in colorectal cancer, advanced adenomas, and normal controls, respectively. On Light -Cycler 480 platform with Resolight/EvaGreen dye, K-ras gene mutation rates were 40%/45% , 31. 4%/34. 2% , and 0%/0% ; whereas in the Rotor-Gene 6000 platform with Resolight/EvaGreen dye, K -ras gene mutation rates were 42. 5%/47. 5% , 34. 2%/37. 1% , and 0%/0% . In each group, there was no significant difference among different operating platforms with different dyes in detection rate of mutations ( P >0. 05 ). The sensitivity of HRM mutation detection in stool was 100%. Conclusion No significant difference in HRM detection rate is found in different operating platforms with different dyes.
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