基于慢病毒载体系统的新型HIV-1基因型耐药检测质控品的制备与评价  

Establishment and evaluation of a new HIV-1 genotypic drug resistance detection quality control using a lentiviral vector system

作　　者：韩頔 张鑫 姚均[1] 刘浩东 赵志艳 韩博学 金聪[1] HAN Di;ZHANG Xin;YAO Jun;LIU Haodong;ZHAO Zhiyan;HAN Boxue;JIN Cong(National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;School of Public Health of Hebei Medical University,Hebei Provincial Key Laboratory of Environment and Population Health,Shijiazhuang 050017,Hebei,China)

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心传染病溯源与智能决策全国重点实验室,北京102206 [2]河北医科大学公共卫生学院,河北省环境与人群健康重点实验室,石家庄050017

出　　处：《中国艾滋病性病》2025年第3期242-248,共7页Chinese Journal of Aids & STD

基　　金：国家重点研发计划项目(2022YFC2305202);北京市自然科学基金-昌平创新联合基金项目(L244069);传染病溯源预警与智能决策全国重点实验室项目。

摘　　要：目的基于HIV-1慢病毒载体(LV)系统制备一种新型HIV-1基因型耐药检测质控品,并对其均一性和稳定性进行评价。方法采用改良的四质粒LV系统包装含有耐药突变K103 N的HIV-1假病毒(PsV)。通过RTPCR和测序方法鉴定携带耐药突变K103 N的HIV-1 PsV包装成功。使用HIV-1阴性血浆作为基质稀释HIV-1 PsV上清液制备HIV-1 PsV耐药检测质控品,并使用反转录数字PCR(RT-dPCR)和耐药位点检测方法对质控品的均一性和稳定性进行评价。结果RT-PCR可扩增出PsV颗粒中插入的含有耐药突变K103 N的pol区基因序列(2648 bp),对扩增产物的测序结果与预期结果一致。从PsV制备的质控品中随机抽取10份使用RT-dPCR检测VL,结果差异无统计学意义(P>0.05);并且使用耐药检测方法均可检出耐药突变K103 N,表明质控品具有良好的均一性。质控品分别在25℃、4℃、-20℃下保存28 d或反复冻融5次,RT-dPCR检测的VL无显著性下降,并且耐药检测均可检出耐药突变K103 N,表明质控品具有良好稳定性。结论基于改良的四质粒LV系统制备的新型HIV-1 PsV耐药质控品具有良好的均一性和稳定性,可以实现对HIV-1基因型耐药检测全过程的质量控制。Objective This study developed a novel quality control material(QCM)for HIV-1 genotypic drug resistance testing using an HIV-1 lentiviral vector(LV)system and evaluated its homogeneity and stability.Methods An improved four-plasmid LV system was used to package the HIV-1 pseudovirus(PsV)carrying the drug resistance mutation,K103 N.Successful packaging of HIV-1 PsV with the K103 N mutation was confirmed via RT-PCR and sequencing.Subsequently,HIV-1 PsV supernatant was diluted with HIV-1-negative plasma as a matrix to obtain the HIV-1PsV drug-resistant QCM.Homogeneity and stability of QCM were evaluated via reverse transcription-digital polymerase chain reaction(RT-dPCR)and drug resistance mutation analyses.Results RT-PCR successfully amplified a 2648-bp pol gene fragment containing the K103 N drug resistance mutation from the PsV particles,and sequencing result of the amplified product matched the expected sequence.Furthermore,10 randomly selected samples of PsV-based QCM were tested for viral load via RT-d PCR.Notably,no statistically significant differences were observed(P>0.05).Additionally,K103 N mutation was consistently detected during drug resistance testing,indicating the excellent homogeneity of QCM.QCM maintained stable viral loads when stored at 25,4,and–20℃for 28 d and subjected to five freeze-thaw cycles,as confirmed via RT-d PCR.K103 N resistance mutation was consistently detected under all conditions,indicating that QCM was stable.Conclusions Overall,this study developed a novel HIV-1 Ps V drug-resistant QCM with excellent homogeneity and stability using an improved four-plasmid LV system and demonstrated its potential for quality control throughout HIV-1 genotypic drug resistance testing.

关 键 词：艾滋病病毒 慢病毒载体 假病毒 耐药检测质控品 反转录数字聚合酶链式反应 

分 类 号：R373.9[医药卫生—病原生物学]