摘要
目的建立一种基于实时荧光定量聚合酶链反应(PCR)方法的1型艾滋病病毒(HIV-1)总脱氧核糖核酸(DNA)定量检测技术。方法构建了同时包含HIV靶基因和内参基因的质粒标准品,设计了涵盖我国主要HIV流行亚型的特异性引物和Taqman水解探针,对引物亚型覆盖面、灵敏度、特异性等相关指标进行了分析,进行了批内和批间重复试验。结果该检测方法所设计的HIV特异性引物和探针序列高度保守,成功检测12种常见及稀有HIV亚型;扩增灵敏度可达4个拷贝/PCR反应管,特异度100%;加入了细胞定量体系,做到了在同一个PCR反应管中既定量了HIV DNA,又定量了细胞数;批内和批间差异性的变异系数均在20%以内。结论该检测方法可用于HIV储藏库检测,评估联合抗病毒治疗的效果,可以用于"窗口期"HIV感染的辅助诊断以及HIV阳性母亲的新生儿HIV感染的诊断。
Objective To establish an ultrasensitive assay to precisely measure the frequency of cells harboring total HIV DNA. Methods HIV-1 LTR gene sequences and CD3 gene sequences were cloned into the PMD20 vector simultaneously, which was used as standards for co-quantification of HIV and cell input. Specific primers and hydrolysis probes covering the main HIV subtypes prevalent in China were designed and aligned with all sequences from the HIV database, and the sensitivity, specificity, coefficient of variation for intra-and inter-assay were evaluated. Results The primers and probes were highly conservative among all HIV-1 subtypes of group M and group N and O. 12 HIV subtypes were successfully detected by using this assay including A, B, b’, C, CRF01AE, CRF07BC, CRF08BC, CRF5501 B, CRF02AG, CRF65cpx, CRF6701 B and CRF01AE/07BC. In addition, a total of 59 HIV-1 positive samples were tested using this assay. Among those, 34(34/35, 97%) samples were positive for HIV-1 DNA with plasma RNA viral load lower than the detection limit(TND), and 24(24/24,100%) samples with plasma RNA viral load positive were all positive for HIV DNA load. The amplification sensitivity of this assay could reach 4 copies per PCR reaction and with a specificity of 100%. The cell input was measured by co-quantification of CD3 gene simultaneously. Inter-assay coefficient of variation and intra-assay coefficient of variation were all below 20%. Conclusion This assay can be used to evaluate the viral reservoir of HIV-1 and the therapeutic effect of cART. It can be also used to help the diagnosis of "window period" HIV infection and babies born to HIV-1-infected mother.
作者
刘志英
袁霖
张欣
陆小凡
刘利锋
计云霞
夏炜
粟斌
吴昊
张彤
LIU Zhiying;YUAN Lin;ZHANG Xin;LU Xiaofan;LIU Lifeng;JI Yunxia;XIA Wei;SU Bin;WU Hao;ZHANG Tong(Beijing You9an Hospital, Capital Medical University , Beijing 100069 , China)
出处
《中国艾滋病性病》
CAS
CSCD
北大核心
2019年第3期222-226,235,共6页
Chinese Journal of Aids & STD
基金
国家"十三五"艾滋病和病毒性肝炎等重大传染病防治课题(2017ZX10202101-004-001
2018ZX10301101-001-001)
北京市卫生系统高层次卫生技术人才(2015-3-102)
北京市艾滋病重点实验室(BZ0089)~~
