摘要
目的使用肿瘤突变负荷(TMB)检测国家参考品,评价基于高通量测序法的肿瘤突变负荷检测试剂盒的TMB检测一致性。方法使用TMB检测试剂盒(可逆末端终止测序法)检测肿瘤突变负荷检测国家参考品中11对来自肿瘤组织永生化的细胞系和配对白细胞永生化的细胞系。首先将DNA打断,进行末端修复、加接头和PCR扩增等步骤后构建文库;然后将文库杂交捕获;最后使用Next⁃Seq550 Dx测序仪进行测序。结果经试剂盒检测,得到TMB⁃1%、TMB⁃2%、TMB⁃5%和TMB⁃10%的TMB检测国家参考品的Panel⁃TMB值。22个TMB⁃5%和TMB⁃10%参考品的标准TMB值落在试剂盒检测计算的WES⁃TMB理论值90%预测区间的比例为90.9%(20/22)。结论研制的国家参考品,能够满足肿瘤突变负荷检测试剂盒的TMB检测一致性评价的要求。
Objective The consistency of tumor mutation burden(TMB)detection was evaluated using the TMB national reference and TMB assay kit based on high throughput sequencing(NGS).Methods 11 pairs of immortalized cell lines from tumor tissues and paired leukocyte immortalized cell lines in national reference materials were tested for TMB using TMB detection kit(reversible terminators for sequencing).First,the library was constructed by shearing DNA,end repair,adapter ligation,PCR amplification and so on.Then the library was hybridized and captured.Finally,the NextSeq550 Dx sequencer was used for sequencing.Results The panel TMB values of TMB-1%,TMB-2%,TMB-5%and TMB-10%of the national reference products were obtained.The proportion of the standard TMB values of TMB-5%and TMB-10%reference products in the 90%prediction interval of the theoretical WES-TMB values calculated by the kit was 90.9%(20/22).Conclusion The developed national reference can meet the requirements of TMB consisten-cy evaluation of the tumor mutation burden detection kit.
作者
孙楠
黄传峰
张文新
黄杰
曲守方
SUN Nan;HUANG Chuanfeng;ZHANG Wenxin;HUANG Jie;QU Shoufang(National Institutes for Food and Drug Control,Beijing,China,100050)
出处
《分子诊断与治疗杂志》
2021年第4期514-517,共4页
Journal of Molecular Diagnostics and Therapy
基金
中国食品药品检定研究院学科带头人培养基金(2019X3)
肿瘤突变负荷(TMB)标准化研究(2019X3)。
