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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。
Objective To establish a real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) for the rapid, sensitive and specific detection of ALK fusion gene in non-small cell lung cancer. Methods Primer Premier 5.0 software was used to design primers and Taqman hydrolysis probes for common fusion mutations V1, V2, V3a and V3b of echinoderm-microtubule-associated protein 4 (EML4) -ALK. Then, the sensitivity, sensitivity and specificity of the established method were further analyzed using the pseudovirions containing EML4-ALK fusion variants V1, V2, V3a and V3b as the research objects. Finally, 50 clinical samples of non-small cell lung cancer were detected by the established method, including 3 ALK-fluorescence in situ hybridization (FISH) (+) samples. Results In the absence of background RNA interference, the established real-time fluorescence quantitative reverse transcription PCR detection sensitivity of up to 10 copies /! The sensitivity was 1% with 500 copies / μl of wild-type background RNA and 0.5% with 5000 copies / μl of wild-type background RNA. For detection specificity, normal human leukocytes and plasma RNA as the research object, no non-specific amplification. The clinical specimens of 50 cases of non-small cell lung cancer were detected in 47 cases of negative specimens were negative, 3 cases of ALK-FISH (+) specimens were positive in 2 cases, 1 case was not detected, the cause was not detected and the sample RNA extraction failed related. Conclusion The real-time fluorescence quantitative RT-PCR method established in this study is a rapid, simple and highly sensitive and specific method for the detection of EML4-ALK fusion gene, which deserves further clinical validation and promotion.