The DNA-based human papilloma virus attacks skin and mucous membranes, causing warts or infections that can lead to precancerous lesions. Clinicians often use methods such as target amplification -- in particular, polymerase chain reaction (PCR) -- to detect such viral DNA. These well-developed techniques offer high efficiency, but they also can yield high numbers of false-positive and false-negative results. In PCR, for example, the presence of small contaminants can lead to false-positive results.Single-molecule spectroscopy does not require amplification, and thus is an attractive alternative for the detection and quantification of viral DNA. Researchers with the Ames Laboratory of the US Department of Energy and with Iowa State University, also in Ames, previously reported an improved single-molecule strategy using a flow system based on fluorescently labeled target DNA molecules passing through a thin fused-silica capillary. This method offered the advantage of a very low detection limit, but also had several limitations; for example, a long collection time for samples with very low concentrations and a need for dilution in samples with high concentrations. Researchers have described the use of surface hybridization with single-molecule spectroscopy for the detection of viral DNA such as human papilloma virus (HPV). They used a dual-probe approach (right) in which the target DNA (HPV) is sandwiched between the second probe (2P-AF, labeled with Alexa Fluor 532) and the surface probe (SP). In single-probe techniques (left) the target DNA itself is labeled with the fluorescent tag. Incorporating the second probe provided a much more rugged fluorescent signal. Images reprinted with permission of Analytical Chemistry. GL = poly(L-lysine)-coated cover glass. Now, in the Nov. 1 issue of Analytical Chemistry, the scientists have reported the use of surface hybridization to overcome these problems. They hybridized target DNA to probes covalently bound on a glass surface -- much as is done in DNA arrays but engineered specifically for single-molecule counting, said co-author Ji-Young Lee. Whereas conventional surface hybridization detection uses only a single probe, the method described by Lee and his colleagues, Jiangwei Li and Edward S. Yeung, incorporates a second probe, and the target DNA is sandwiched between this probe and the surface. Thus, the dual-probe technique provides a rugged fluorescence signal, Lee said, while not interfering with the hybridization of the target sequence. Images acquired using dual-probe hybridization detection demonstrate the efficacy of the technique. The panels here represent samples with 0 (A), 10–4 (B), 10–3 (C), 10–2 (D), 10 (E) and 1 percent human papilloma virus DNA. Each dot in the images reflects one probe molecule. To achieve better hybridization and reproducibility, they used 50- to 100-bp sequences of human papilloma virus DNA as probes. They had first tested quantum dots, aiming for a bright fluorescence tag without compromising the hybridization efficiency. Lee noted that the detection efficiency was not high enough, however. In addition, the DNA was sheared easily in the washing steps, and there was reduced fluorescence from the quantum dots after overnight hybridization. Therefore, the researchers used Alexa Fluor 532 covalently bound to a DNA fragment. They knew that, under the conditions that they used, DNA would coil randomly on the slide surface after hybridization, leading to less shearing and more robust performance of the tag throughout the hybridization step.They validated the technique first by testing the surface hybridization with the single- and dual-probe methods using purified commercial DNA, one normal cell line and two cell lines with known human papilloma virus infection. Then they applied the dual-probe strategy to healthy human cervix cells acquired through conventional Pap test sampling.In each case, the investigators positioned the slides atop a right-angle fused-silica prism made by Melles Griot (now CVI Melles Griot) of Carlsbad, Calif., separated by only a layer of immersion oil. A 532-nm solid-state continuous-wave laser made by Uniphase Corp. (now JDSU) provided excitation, with the beam modulated by a mechanical shutter and driver synchronized to a Princeton Instruments intensified CCD camera. A 100×, 1.3-NA objective lens made by Carl Zeiss collected the fluorescence from individual hybridized molecules, which was then imaged by the camera. Two 532-nm long-pass edge filters positioned between the objective and the camera eliminated scattering from the excitation beam.The researchers determined the human papilloma virus contents for the cell line extracts based on the average molecule counts for each image, and found that all were in good agreement with known viral loads. The Pap smear results showed good agreement as well, and furthermore confirmed the compatibility of the technique with the most widely used Pap smear sample collection methods.The technique could provide a robust alternative for clinical screening and quantification of viral DNA in cells. Because no amplification is needed, quantification is far less complicated. Also, the method offers a detection limit as low as approximately 1.44 copies per cell and a wide linear dynamic range of greater than five orders of magnitude, which covers the clinically important range of viral content from early infection to the cancer-forming stage, Lee said. Furthermore, it allows higher throughput than the flow system the researchers previously used for single-virus detection, without compromising detection performance.The technique is not limited to the detection of human papilloma virus. By modifying the probe DNA design, it can be used to screen other targets, including HIV. The researchers are working to apply the technique for the detection of multiple forms of the same virus, or of multiple viruses, by using different color labels on the second probe.According to Yeung, who is the principal investigator, some engineering will be required to get the system into a package that can be placed in clinical laboratories.