人类白细胞抗原B27核酸检测国家参考品的建立  

Establishment of the National reference materials for Human Leukocyte Antigen B27 Nucleic Acid Detection

作　　者：胡泽斌[1] 高飞[1] 孙楠[1] 孙彬裕[1] 李丽莉[1] 孙晶 曲守方[1] 黄杰[1] HU Zebin;GAO Fei;SUN Nan;SUN Binyu;LI Lili;SUN Jing;QU Shoufang;HUANG Jie(Division of In Vitro Diagnostic Reagents,National Institute for Food and Drug Control,Beijing,China,100050)

机构地区：[1]中国食品药品检定研究院体外诊断试剂检定所,北京100050

出　　处：《分子诊断与治疗杂志》2020年第10期1285-1288,1293,共5页Journal of Molecular Diagnostics and Therapy

基　　金：生殖健康及重大出生缺陷防控研究(2016YFC1000300)。

摘　　要：目的建立人类白细胞抗原B27核酸检测国家参考品。方法采集HLA-B27阳性和阴性志愿者的新鲜外周血,经EB病毒转化,培养建立永生化细胞系。提取细胞基因组DNA,制备成国家参考品并经高通量测序验证。用4家协作标定单位的HLA-B27核酸检测试剂,采用荧光PCR法、PCR-SSP法、SBT测序法对国家参考品进行定值,并进行国家参考品的复溶(在-20℃和25℃之间反复冻融3次)稳定性和均匀性研究。结果成功制备HLA-B27国家参考品,包含3例HLA-B27阳性DNA样本和7例HLA-B27阴性DNA样本。经荧光PCR法、PCR-SSP法、SBT测序法协作标定,参考品定值准确。参考品均匀性结果一致,参考品复溶3次后仍然稳定。结论人类白细胞抗原B27核酸检测国家参考品的各项指标均符合要求,可用于人类白细胞抗原B27核酸检测试剂盒的性能评价。Objective To establish a national reference material for HLA-B27 nucleic acid detection.Methods Fresh peripheral blood of HLA-B27 positive and negative volunteers was collected,transformed with EB virus,and cultured to establish immortalized cell lines.The genomic DNA was extracted from the cells,and prepared for the national reference material which was verified by the next generation sequencing technology.Moreover,the accuracy of the national reference was verified by fluorescence PCR,PCR-SSP method and SBT sequencing method,with HLA-B27 nucleic acid detection reagents from four collaborative manufacturers.The stability after 3 cycles of freezing and thawing at-20℃and 25℃and homogeneity of the national reference materials were studied as well.Results The national reference materials of HLA-B27 were successfully prepared,including 3 HLA-B27 positive DNA samples and 7 HLA-B27 negative DNA samples.The reference materials were accurately valued by SBT sequencing and other methods,and the homogeneity of reference material was consistent.The reference material is stable after 3 cycles of freezing-thawing,which met the requirements of the national reference material.Conclusion All indexes of the national reference material for human leukocyte antigen B27 nucleic acid detection meet the requirements and can be used for the performance evaluation of human leukocyte antigen B27 nucleic acid detection kit.

关 键 词：人类白细胞抗原 B27 SBT测序法 国家参考品 协作标定 

分 类 号：R440[医药卫生—诊断学]