【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。更多还原

【Abstract】 Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay( qRT-PCR) for the rapid,sensitive,and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase( EML4-ALK) fusion genes in non-small cell lung cancer. Methods Thespecific primers for the four variants of EML4-ALK fusion genes( V1,V2,V3 a,and V3b) and Taqman fluorescence 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,V3 a,and V3b) as the study objects,we further analyzed the lower limit,sensitivity,and specificity of this method. Finally,50 clinical samples,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 / !l 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 / !l 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 volunteers. 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 settings.更多还原