Single-Molecule Detection of Surface-Hybridized Human Papilloma Virus DNA for Quantitative Clinical Screening

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• 作者：Ji-Young Lee ; Jiangwei Li ; Edward S. Yeung
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：November 1, 2007
• 年：2007
• 卷：79
• 期：21
• 页码：8083 - 8089
• 全文大小：383K
• 年卷期：v.79,no.21(November 1, 2007)
• ISSN：1520-6882

文摘

We present an improved method to quantify viral DNA inhuman cells at the single- molecule level. Human papilloma virus (HPV)-16 DNA was hybridized to probes thatwere covalently bound to a glass surface and detected witha single-molecule imaging system. In the single-probemode, the whole genome and target DNA were fluorescently labeled before hybridization. In the dual-probemode, a second probe was introduced that has a fluorescently labeled 1-kb DNA strand connected to the 50-ntprobe sequence. With the single-probe method, the detection limit was 0.7 copy/cell, which was similar to thatreported in a flow system earlier. With the dual-probemethod, the linear dynamic range covers 1.44-7000copies/cell, which is typical of early infection to near-cancer stages. Both methods were applied to cell linesamples with known HPV-16 infection, and the resultshowed a good match with the reported viral load. DNAfrom cervical cells, collected with the Pap smear samplingmethod, was spiked with HPV-16 DNA and submitted tothis assay to show compatibility with conventional sampling methods. The dual-probe method was further testedwith a crudely prepared sample. The cells were heat lyzedand spun down, and the supernatant was immediatelysubmitted to hybridization. Even with reduced hybridization efficiency caused by the interference of cellularmaterials, we were still able to differentiate infected cellswith 600 copies/cell from healthy cells.