Single-molecule detection of surface-hybridized human papillorna virus DNA for quantitative clinical screening
Single-molecule detection of surface-hybridized human papillorna virus DNA for quantitative clinical screening
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DOI：
10.1021/ac071159y
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Yeung, Edward S.
中科院分区：
文献类型：
--
作者：
Lee, Ji-Young;Li, Jiangwei;Yeung, Edward S.
我们提出了一种改进的方法，在单分子水平上对人类细胞中的病毒DNA进行量化。将人乳头瘤病毒(HPV)-16DNA与共价结合在玻璃表面的探针杂交，并用单分子成像系统进行检测。在单探针模式中，在杂交前对整个基因组和目标DNA进行荧光标记。在双探针模式中，引入了第二个探针，该探针具有连接到50-nT探针序列的荧光标记的1-kb DNA链。单探针法的检测下限为0.7拷贝/细胞，与先前在流动体系中报道的结果相似。双探针法的线性动态范围为1.44-7000拷贝/细胞，这是早期感染到近癌阶段的典型特征。将这两种方法应用于已知HPV-16感染的细胞系样本，结果显示与报道的病毒载量有很好的匹配。用巴氏涂片取样法采集的宫颈细胞DNA被加入HPV-16DNA，并提交给该检测，以显示与传统采样方法的兼容性：双探针方法进一步用粗制的样本进行测试。这些细胞被热裂解并旋转下来，上清液立即提交给杂交。即使在细胞材料干扰导致杂交效率降低的情况下，我们仍然能够从健康细胞中区分出600拷贝/细胞的感染细胞。
We present an improved method to quantify viral DNA in human cells at the single-molecule level. Human papilloma virus (HPV)-16 DNA was hybridized to probes that were covalently bound to a glass surface and detected with a single-molecule imaging system. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization. In the dual-probe mode, a second probe was introduced that has a fluorescently labeled 1-kb DNA strand connected to the 50-nt probe sequence. With the single-probe method, the detection limit was 0.7 copy/cell, which was similar to that reported in a flow system earlier. With the dual-probe method, the linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. Both methods were applied to cell line samples with known HPV-16 infection, and the result showed a good match with the reported viral load. DNA from cervical cells, collected with the Pap smear sampling method, was spiked with HPV-16 DNA and submitted to this assay to show compatibility with conventional sampling methods: The dual-probe method was further tested with a crudely prepared sample. The cells were heat lyzed and spun down, and the supernatant was immediately submitted to hybridization. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells with 600 copies/cell from healthy cells.