二代测序技术在用于脊髓灰质炎病毒分离的细胞系交叉污染鉴定中的应用  

Application of next generation sequencing(NGS)for identifying cross-contamination of cells for poliovirus isolation

作　　者：王雪 康露 龚成[1] 李爱华[1] 李茂中[1] 张合润[1] 黄芳[1] 罗明[1] Wang Xue;Kang Lu;Gong Cheng;Li Aihua;Li Maozhong;Zhang Herun;Huang Fang;Luo Ming(Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]北京市疾病预防控制中心,北京100013

出　　处：《中国疫苗和免疫》2022年第5期563-568,共6页Chinese Journal of Vaccines and Immunization

基　　金：首都卫生发展科研专项(2021-1G-3015)。

摘　　要：目的应用二代测序(Next generation sequencing,NGS)技术鉴定用于脊髓灰质炎(脊灰)病毒分离的RD和L20B细胞种属来源,以判断细胞间是否存在交叉污染。方法选用新复苏的RD和L20B细胞作为对照组,传代多代的RD和L20B细胞作为实验组,观察在接种两种非脊灰肠道病毒株后的细胞形态和细胞病变(Cytopathic effect,CPE),检测细胞的支原体和细胞对脊灰病毒的敏感性;利用NGS技术对细胞进行高通量测序和比对,以鉴定细胞的种属来源、识别交叉污染。结果与对照组RD细胞相比,实验组RD细胞形态出现膨胀、条柱形等变化,实验组L20B细胞形态相似;接种两种非脊灰肠道病毒毒株后,对照组RD细胞发生CPE而L20B细胞未发生CPE,实验组两种细胞均发生CPE。对照组和实验组两种细胞支原体检测均阴性,且敏感性结果均处于正常范围内。经NGS技术鉴定,实验组RD和L20B细胞均属于人源细胞,L20B细胞的种属来源与预期不符。结论实验组RD和L20B细胞间存在交叉污染;NGS技术能够快速、准确、全面地鉴定细胞系的种属来源,及时识别细胞间交叉污染,可进一步推动脊灰病毒分离用细胞系质量控制工作。Objective To identify species of RD and L20B cells for poliovirus isolation using the next generation sequencing(NGS)technique and detect intercellular cross-contamination.Methods We selected newly revived RD and L20B cells as a control group and multi-passage RD and L20B cells as an experimental group,observed their morphological characteristics and cytopathic effects(CPE)after inoculation with two non-poliovirus enterovirus strains,and tested for mycoplasma in cells and the sensitivity of cells to poliovirus.We used the NGS technique to conduct high-throughput sequencing and alignment of cells and identify cell species and cross-contamination.Results Compared with RD cells in the control group,RD cells in the experimental group showed swelling and columnar morphology while L20B cells were similar in morphology.After inoculation with two non-poliovirus enterovirus strains,CPE occurred in RD cells other than L20B cells of the control group,while CPE occurred in both cells of the experimental group.The two cells in both groups were negative for mycoplasma,with sensitivity in the normal range.The NGS technique showed that RD and L20B cells in the experimental group were identified as human cells,while the species of L20B cells was not.Conclusions Cross-contamination between RD and L20B cells existed in the experimental group.The NGS technique may quickly and accurately be able to comprehensively identify the species of cell lines and intercellular cross-contamination and improve quality control of cell lines for poliovirus isolation.

关 键 词：脊髓灰质炎病毒 RD细胞 L20B细胞 种属 细胞间交叉污染 细胞系质量控制 二代测序技术 

分 类 号：R331[医药卫生—人体生理学] R183.4[医药卫生—基础医学]