Single-molecule detection of surface-hybridized human papilloma virus DNA for quantitative clinical screening.

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.