基于连接酶链式反应的EML4-ALK融合基因分型检测

Typing detection of EML4-ALK fusion gene based on ligase chain reaction

针对棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶(EML4-ALK)融合基因变异体V1、V2、V3a、V3b设计引物、探针,优化连接探针长度及其浓度,建立基于连接酶链式反应的qPCR法检测方案,并对该方案的特异性、检出限、抗干扰性进行验证。结果表明:基于连接酶链式反应的qPCR法能对EML4-ALK融合基因进行有效分型;连接探针长度在30 nt(nucleotide)时检测灵敏度、特异性最高;连接探针终浓度在0.1~1.0 nmol/L较为合适;设计的引物、探针特异性好,只扩增靶RNA,对其他RNA无扩增;对EML4-ALK融合基因变异体V1、V2的检出限为10 copies/μL,变异体V3a、V3b的检出限为100 copies/μL;在EML4-ALK融合基因RNA标准品检测过程中加入100 ng的肺癌样本RNA作为干扰,检测结果未受到明显干扰。

Primers and probes were designed for echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase(EML4-ALK)fusion gene variants V1,V2,V3a and V3b,and the ligase chain reaction-based qPCR assay protocol was established by optimizing the length and concentration of ligation probes,and the specificity,detection limit and interference resistance of the protocol were verified.The results show that the ligase chain reaction-based qPCR method can effectively type the EML4-ALK fusion gene;the detection sensitivity and specificity are highest when the length of the ligation probe is 30 nt(nucleotide);a final concentration of 0.1-1.0 nmol/L is appropriate for the ligation probe;the designed ligation probes,primers and TaqMan probes are specific and amplified only the target RNA without amplification of other RNAs;the detection limits of EML4-ALK fusion gene variants V1 and V2 are 10 copies/μL,and the detection limits of variants V3a and V3b are 100 copies/μL;100 ng of RNA from lung cancer samples are added as interference during the detection of EML4-ALK fusion gene RNA standards,and the detection results are not significantly interfered.