摘要
目的 验证实验室自建人类免疫缺陷病毒1型(HIV-1)整合酶基因序列分析方法。该方法可用于评估HIV-1整合酶区段基因型耐药水平。方法 根据世界卫生组织自建基因序列分析方法验证的建议,从20份HIV-1阳性样本中提取RNA,扩增HIV-1整合酶区基因片段,并测序。通过与病毒质量保证(VQA)共识进行比对,评估实验室自建的HIV-1整合酶基因序列分析方案的准确性,通过扩增成功率评估其灵敏度,通过同一样本的重复检测结果评估其精密度和重现性。结果 20份样本与VQA共识的核苷酸一致率均>98%;10个高病毒载量(>10 000拷贝·mL^(-1))样本和5个低病毒载量(1 000~5 000拷贝·mL^(-1))样本的扩增成功率均为100%;4个样本的同批次5复孔和5个样本5次检测的结果均符合90%的样本配对比较核苷酸一致率>98%的要求。结论 该HIV-1整合酶基因序列分析方法的准确性、灵敏度、精密度和重现性均符合要求,适用于HIV-1整合酶基因序列分析。
Objective To validate laboratory-developed human immunodeficiency virus-1(HIV-1)integrase genotyping sequence assay which can be applied to assess the level of genotyping drug resistance to HIV-1 integrase inhibitors.Methods According to the World Health Organization self-developed gene sequence analysis method validation recommendation,RNA was extracted from 20 HIV-1 positive samples,and the gene fragments of HIV-1 integrase region were amplified and sequenced.The accuracy of laboratory-developed HIV-1 integrase genotyping sequence assay was evaluated by comparison with virus quality assurance(VQA)consensus.Amplification sensitivity was evaluated by amplification success rate.Precision and reproducibility were evaluated by repeated tests of the same sample.Results All the 20 samples had a nucleotide agreement rate of>98%with VQA consensus.The multi-well amplification rate of 10 high viral load samples(>10000 copies·mL^(-1))and 5 low viral load samples(1000-5000 copies·mL^(-1))was 100%.The results of 5 complex wells of 4 samples of the same batch,as well as 5 assays of 5 samples,met the requirement of nucleotide agreement rate of>98%in 90%sample pairwise comparison.Conclusions The accuracy,amplification sensitivity,precision and reproducibility of this assay meet all the requirements and are suitable for HIV-1 integrase genotyping.
作者
王绪琴
林倩茹
冯琬清
董原
郁晓磊
刘长河
宁镇
沈鑫
潘启超
林怡
WANG Xuqin;LIN Qianru;FENG Wanqing;DONG Yuan;YU Xiaolei;LIU Changhe;NING Zhen;SHEN Xin;PAN Qichao;LIN Yi(Shanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China)
出处
《检验医学》
CAS
2024年第4期369-375,共7页
Laboratory Medicine
基金
上海市自然科学基金项目(21ZR1459900)
上海市加强公共卫生体系建设三年行动计划(2023-2025)重点学科项目(GWVI-11.1-05)。
关键词
人类免疫缺陷病毒1型
整合酶基因序列分析
基因型耐药检测
Human immunodeficiency virus-1
Integrase genotyping sequence assay
Genotyping drug resistance determination
