纳米孔测序技术检测辐射损伤基因表达方法的建立

An amplicon sequencing assay based on nanopore sequencing technology for detecting the expressions of radiation damage genes

目的 建立纳米孔测序技术检测辐射损伤基因表达的方法。方法 根据文献设计并制备了3种21个辐射损伤基因引物组合,提取健康志愿者外周血总RNA,逆转录为cDNA,经数字PCR(dPCR)定量上述辐射损伤基因,并根据结果筛选最佳的引物组合。采集5例初次放疗肿瘤患者放疗前、后的新鲜外周血样本并提取RNA,经逆转录和辐射损伤基因引物多重PCR,应用纳米孔测序技术对多重引物PCR产物进行测序,同时对外周血RNA进行二代测序,并分析两种测序方法对受照前后外周血中辐射损伤基因表达水平变化是否具有一致性。结果 筛选出与dPCR结果拟合优度最佳的引物组合;在纳米孔测序开始后第1、2、3小时所产生的数据量显著一致(P<0.01),1 h所产生的测序结果即可满足21个辐射损伤基因表达水平分析所需数据量,达到快速检测目的;纳米孔测序与二代测序数据对比显示,5名患者放疗后表达谱均有相同变化趋势;两种手段所获辐射损伤基因表达水平变化一致(P<0.05)。结论 建立了基于纳米孔测序技术辐射损伤相关基因表达谱检测方法,可用于辐射损伤快速现场评估。

Objective To establish a method for detecting the expression profile of radiation damage genes based on nanopore sequencing technology. Methods Three sets of 21 primer combinations of radiation damage genes were designed and prepared according to literature. Total RNA was extracted from the peripheral blood of healthy volunteers and reversely transcribed into cDNA. The above radiation damage genes were quantified by digital PCR(dPCR),and the optimal primer sets were chosen according to the results. Fresh peripheral blood samples of 5 tumor patients before and after primary radiotherapy were collected and the RNA was extracted. Then,RNA reverse transcription and multiplex PCR targeting radiation damage genes were performed. Multiprimers PCR products were sequenced by nanopore sequencing,and peripheral blood RNA was sequenced by the second generation,the consistency of the two sequencing methods on the expression level of radiation damage genes in peripheral blood before and after exposure was analyzed. Results The primer set with the least bias to dPCR results was selected. The amount of data generated in the first,second and third hours after nanopore sequencing had significant consistency(P<0.01). A comparison of nanopore sequencing and secondgeneration sequencing data showed that the expression profiles of the 5 patients changes in the same way after radiotherapy.The changes in the expression levels of radiation damage genes obtained by the two methods were significantly consistent(P<0.05),which verified the reliability of this method. Conclusion A method for detection of expressions of radiation damage genes based on nanopore sequencing technology is established,which can be used for rapid assessment of on-site radiation injuries.