中国医学科学院学报2016，Vol.38Issue(6)：643-649,7.DOI:10.3881/j.issn.1000-503X.2016.06.004
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
Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method
赵静 1赵金银 2陈志霞 1钟巍 1李龙芸 1刘利成 2胡小许 2陈唯军 2王孟昭1
作者信息
- 1. 中国医学科学院 北京协和医学院 北京协和医院呼吸内科，北京100730
- 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，北京100029
- 折叠
摘要
Abstract
Objective To establish a real-time quantitative reverse transcription polymerase chain reac-tion assay ( qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated pro-tein like 4-anaplastic lymphoma kinase ( EML4-ALK) fusion genes in non-small cell lung cancer. Methods The <br> specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluores-cence probes for the detection of the target sequences were carefully designed by the Primer Premier 5. 0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study ob-jects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical sam-ples, including 3 ALK-fluorescence in situ hybridization ( FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/ml if no interference of background RNA existed. Regarding the method’ s sensitivity, the detection resolution was as high as 1% and 0. 5% in the background of 500 and 5000 copies/ml wild-type ALK gene, respectively. Regarding the method’ s specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volun-teers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical set-tings.关键词
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/实时荧光定量逆转录多聚酶链反应Key words
non-small cell lung cancer/echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase fusion gene/real-time quantitative reverse transcription polymerase chain reaction分类
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赵静,赵金银,陈志霞,钟巍,李龙芸,刘利成,胡小许,陈唯军,王孟昭..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[J].中国医学科学院学报,2016,38(6):643-649,7.
