基于CRISPR/Cas13a的EML4-ALK融合基因检测方法的建立

Establishment of a novel method in detecting EML4-ALK fusion gene based on CRISPR/Cas13a

[目的]构建基于CRISPR/Cas13a蛋白的SHERLOCK技术平台,并将其应用于肺癌EML4-ALK融合基因的检测。[方法]构建EML4-ALK融合基因的质粒标准品;利用RPA和PCR技术分别扩增目的基因后进行SHERLOCK检测并比对结果,比较基于RPA和PCR扩增的SHERLOCK检测EML4-ALK融合基因的灵敏度,评估SHERLOCK检测目的基因及多种非目标基因的特异性。[结果]成功构建EML4-ALK融合基因表达质粒标准品;基于RPA和PCR扩增的SHERLOCK检测EML4-ALK融合基因的相对荧光值无显著差异,最低检测下限为10 copies/μL,且在5 min内荧光达到平台期;SHERLOCK技术仅对EML4-ALK融合基因有阳性检测信号,对EML4、ALK、EGFR T790M和EGFR L858R基因无阳性检测信号。[结论]基于SHERLOCK技术的EML4-ALK融合基因检测方法具有特异性强、灵敏度高、检测时间短等优势,有望为肺癌EML4-ALK融合基因的早期诊断提供重要依据。

[Objective] The SHERLOCK technology platform based on CRISPR/Cas13a protein was constructed and applied to the detection of EML4-ALK fusion gene in lung cancer.[Method]Constructing plasmid standard of EML4-ALK fusion gene.After the target genes were amplified by RPA and PCR techniques, SHERLOCK detection was performed to compare the sensitivity of SHERLOCK based on RPA and PCR amplification and to evaluate the specificity of SHERLOCK detection of target genes and various non-target genes.[Result]The standard EML4-ALK fusion gene expression plasmid was successfully constructed.SHERLOCK based on RPA and PCR amplification showed no significant difference in the relative fluorescence value of EML4-ALK fusion gene, with a minimum detection limit of 10 copies/μL and a plateau in fluorescence within 5 min.SHERLOCK technique only had positive detection signal for EML4-ALK fusion genes, but had no positive detection signal for EML4-ALK,EGFR T790M and EGFR L858R genes.[Conclusion] The EML4-ALK fusion gene detection method based on SHERLOCK technology has the advantages of strong specificity, high sensitivity and short detection time, and is expected to provide an important basis for the early diagnosis of EML4-ALK fusion gene in lung cancer in lung cancer.