建立实时荧光定量逆转录PCR方法检测非小细胞肺癌棘皮动物微管相关蛋白4-间变淋巴瘤激酶融合基因

发布时间：2018-01-13 05:32

本文关键词：建立实时荧光定量逆转录PCR方法检测非小细胞肺癌棘皮动物微管相关蛋白4-间变淋巴瘤激酶融合基因　出处：《中国医学科学院学报》2016年06期 　论文类型：期刊论文

【摘要】：目的建立一种实时荧光定量逆转录PCR方法,对非小细胞肺癌间变淋巴瘤激酶(ALK)融合基因进行快速、敏感和特异检测。方法首先,应用Primer Premier 5.0软件,针对棘皮动物微管相关蛋白4(EML4)-ALK常见融合变异V1、V2、V3a和V3b设计引物和Taqman水解探针。然后,以包含EML4-ALK融合变异V1、V2、V3a和V3b的假病毒颗粒为研究对象,进一步分析所建立方法的灵敏度、敏感性和特异性。最后,用所建立的方法检测50例非小细胞肺癌临床标本,其中包含3例ALK-荧光原位杂交(FISH)(+)样本。结果在无背景RNA干扰情况下,建立的实时荧光定量逆转录PCR方法检测灵敏度高达10拷贝/!l。在500拷贝/!l的野生型背景RNA下,其敏感性达1%,在5000拷贝/!l的野生型背景RNA下,其敏感性达0.5%。对于检测特异性,以正常人白细胞及血浆RNA为研究对象,均未见非特异性扩增。对50例非小细胞肺癌临床标本进行检测,47例阴性标本检测均为阴性,3例ALK-FISH(+)标本2例检测阳性,1例未检出,未检出原因与标本RNA提取失败有关。结论本研究建立的实时荧光定量逆转录PCR方法是一种快速、简便以及具有高灵敏度和特异性的EML4-ALK融合基因检测方法,值得在临床进一步验证和推广。
[Abstract]:Objective to establish a real-time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) method for rapid, sensitive and specific detection of ALKs fusion gene in non-small cell lung cancer. Using Primer Premier 5.0 software, the common fusion variant V1 / V2 of EML4- ALK was used for echinoderms microtubule-associated protein 4ML-4- ALK. Primers and Taqman hydrolysis probes were designed for V3a and V3b. Then, pseudoviral particles containing EML4-ALK fusion mutation V1 / V2V3a and V3b were selected as the study objects. The sensitivity, sensitivity and specificity of the established method were further analyzed. Finally, 50 clinical specimens of non-small cell lung cancer were detected by the established method. Three samples of ALK-fluorescence in situ hybridization (fish) were included. The results were without background RNA interference. The sensitivity of the real-time fluorescent quantitative reverse transcriptase (PCR) assay is as high as 10 copy / s! In 500 copies! L with a wild-type background RNA, its sensitivity is as high as 1, 000 copies / /! Under wild-type background RNA, the sensitivity was 0.5%. For detection specificity, normal human white blood cells and plasma RNA were studied. No nonspecific amplification was found in all of the 50 non-small cell lung cancer clinical specimens. All the 47 negative specimens were negative and 3 were ALK-FISH(). 2 were positive for ALK-FISH(). One case was not detected and the reason of non-detection was related to the failure of RNA extraction. Conclusion the real-time fluorescent quantitative reverse transcriptase (PCR) method established in this study is a rapid method. A simple and highly sensitive and specific method for detection of EML4-ALK fusion gene is worthy of further validation and promotion in clinical practice.
【作者单位】：中国医学科学院北京协和医学院北京协和医院呼吸内科;中国科学院北京基因组研究所基因组科学与信息重点实验室;
【分类号】：R734.2
【正文快照】： 2中国科学院北京基因组研究所基因组科学与信息重点实验室,北京1000291Department of Respiratory Medicine,PUMC Hospital,CAMS and PUMC,Beijing 100730,China2Key Laboratory of Genome Sciences and Information,Beijing Institute of Genomics,Chinese Academy of Scienc

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