总HIV-1 DNA定量检测技术的建立
|
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, CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01 B, CRF02_AG, CRF65_cpx, CRF67_01 B and CRF01_AE/07_BC. 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.
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, CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01 B, CRF02_AG, CRF65_cpx, CRF67_01 B and CRF01_AE/07_BC. 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.
引文
[1] 中国疾病预防控制中心性病艾滋病预防控制中心性病控制中心. 2018年5月全国艾滋病性病疫情[J]. 中国艾滋病性病, 2018,24(7):645.
[2] D Persaud, Patel K, Karalius B, et al. Influence of age at virologic control on peripheral blood human immunodeficiency virus reservoir size and serostatus in perinatally infected adolescents[J]. JAMA Pediatr, 2014,168(12):1138-1146.
[3] Laurent Hocqueloux, Véronique Avettand-Fèno?l, Jade Ghosn,et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications[J]. Clin Microbiol Rev, 2016,29(4):859-880.
[4] 李海英, 刘志英, 张彤,等. 北京市部分男男同性恋人群中HIV-1分子流行病学研究[J]. 首都医科大学学报, 2009,30(5): 616-620.
[5] 梁淑家,李天一, 陈立力, 等. 广西首例HIV-1G亚型毒株的发现和近似全长基因组序列分析[J]. 军事医学, 2012, 36(3):192-195.
[6] 钟平,汪宁. 中国HIV分子流行病学30年[J]. 中华流行病学杂志,2015,6(6):541,546.
[7] Han XX,Yutaka Takebe, Zhang WQ, et al.A Large-scale Survey of CRF55_01B from Men-Who-Have-Sex-with-Men in China: implying the Evolutionary History and Public Health Impact[J]. Sci Rep,2015(5):18147.
[8] Li HP, Bao ZY, Wang XL, et al. Phylogenetic Analysis of HIV-1 CRF65_CPX Reveals Yunnan Province Is Still a Source Contributing to the Spread of HIV-1 in China[J]. Journal of acquired immune deficiency syndromes,2015,70(3):e120-e122.
[9] Ning CY, Li XG, He X,et al. Genome sequences of a novel HIV-1 circulating recombinant form (CRF61_BC) identified among heterosexuals in China[J]. Genome Announc, 2013,1(3):e00326-13.
[10] S Eriksson, Graf EH, Dahl V, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies[J]. PLoS Pathog, 2013,9(2):e1003174.
[11] AM Crooks, Bateson R, Cope AB, et al. Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication Strategies[J]. Infect Dis,2015,212(9):1361-1365.
[12] F Hong, Aga E, Cillo AR, et al. Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA[J]. Clin Microbiol, 2016,54(4):902-911.
[13] Lada SM, Strain MC, Luong T, et al. Highly Precise Measurement of HIV DNA by Droplet Digital PCR[J]. PLoS One,2013,8(4):1-8.
[14] KJ Bosman, Nijhuis M, van Ham PM, et al. Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir[J]. Sci Rep, 2015(5):13811.
[15] Kajdas J, Siliciano JD, Finzi D,et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells[J]. Nat Med, 2003,9(6):727-728.
[2] D Persaud, Patel K, Karalius B, et al. Influence of age at virologic control on peripheral blood human immunodeficiency virus reservoir size and serostatus in perinatally infected adolescents[J]. JAMA Pediatr, 2014,168(12):1138-1146.
[3] Laurent Hocqueloux, Véronique Avettand-Fèno?l, Jade Ghosn,et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications[J]. Clin Microbiol Rev, 2016,29(4):859-880.
[4] 李海英, 刘志英, 张彤,等. 北京市部分男男同性恋人群中HIV-1分子流行病学研究[J]. 首都医科大学学报, 2009,30(5): 616-620.
[5] 梁淑家,李天一, 陈立力, 等. 广西首例HIV-1G亚型毒株的发现和近似全长基因组序列分析[J]. 军事医学, 2012, 36(3):192-195.
[6] 钟平,汪宁. 中国HIV分子流行病学30年[J]. 中华流行病学杂志,2015,6(6):541,546.
[7] Han XX,Yutaka Takebe, Zhang WQ, et al.A Large-scale Survey of CRF55_01B from Men-Who-Have-Sex-with-Men in China: implying the Evolutionary History and Public Health Impact[J]. Sci Rep,2015(5):18147.
[8] Li HP, Bao ZY, Wang XL, et al. Phylogenetic Analysis of HIV-1 CRF65_CPX Reveals Yunnan Province Is Still a Source Contributing to the Spread of HIV-1 in China[J]. Journal of acquired immune deficiency syndromes,2015,70(3):e120-e122.
[9] Ning CY, Li XG, He X,et al. Genome sequences of a novel HIV-1 circulating recombinant form (CRF61_BC) identified among heterosexuals in China[J]. Genome Announc, 2013,1(3):e00326-13.
[10] S Eriksson, Graf EH, Dahl V, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies[J]. PLoS Pathog, 2013,9(2):e1003174.
[11] AM Crooks, Bateson R, Cope AB, et al. Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication Strategies[J]. Infect Dis,2015,212(9):1361-1365.
[12] F Hong, Aga E, Cillo AR, et al. Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA[J]. Clin Microbiol, 2016,54(4):902-911.
[13] Lada SM, Strain MC, Luong T, et al. Highly Precise Measurement of HIV DNA by Droplet Digital PCR[J]. PLoS One,2013,8(4):1-8.
[14] KJ Bosman, Nijhuis M, van Ham PM, et al. Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir[J]. Sci Rep, 2015(5):13811.
[15] Kajdas J, Siliciano JD, Finzi D,et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells[J]. Nat Med, 2003,9(6):727-728.