Detection of integrated HIV-1 genome in epithelial cells by qPCR.

aC8166 cells express CXCR4 and were used for X4 viral infections only.bNP2-R5 cells express CCR5 and were used for R5 viral infections only.+, Integrated HIV-1 product detected (cycle threshold detection in brackets).ND, Integrated HIV-1 product not detected.Real-time PCR assay to detect integrated HIV-DNA using HIV-1 LTR and human Alu-specific primers and a U5 specific probe [92]. TR146, FaDu, A431 and PM-1 (control) cells were exposed to YU2 (R5) or LAI (X4) virus for 48 h at 37°C. DNA was extracted, digested with DpnI to degrade any plasmid DNA contaminant and analysed by real-time PCR.

aC8166细胞表达C-X-C趋化因子受体4（CXCR4），仅用于X4嗜性病毒感染实验。bNP2-R5细胞表达C-C趋化因子受体5（CCR5），仅用于R5嗜性病毒感染实验。“+”代表检测到整合型HIV-1产物，括号内标注循环阈值检测值；“ND”代表未检测到整合型HIV-1产物。本实验采用实时荧光定量PCR（real-time PCR）技术，以HIV-1长末端重复序列（LTR）、人类Alu特异性引物及U5特异性探针为检测体系，用于扩增检测整合型HIV-DNA[92]。将TR146、FaDu、A431及作为对照的PM-1细胞置于37℃条件下，分别与YU2（R5型）或LAI（X4型）病毒共培养48小时。随后提取细胞DNA，经DpnI酶切以降解残留的质粒DNA污染物，再通过实时荧光定量PCR完成分析。