Typing detection of EML4-ALK fusion gene based on ligase chain reaction
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.
万方数据
维普
