Hybridization assay performed at ambient temperature for typing high-risk human papillomaviruses.

BACKGROUND: Human papillomavirus (HPV) infection with oncogenic types is a prerequisite for cervical cancer development. HPV typing is required in the management of pre-cancerous lesions, epidemiological studies, and vaccination trials. None of the available HPV assays are satisfactory for routine diagnosis. OBJECTIVES: In order to develop an assay for clinically relevant HPV types, we generated HPV probes using in vitro selection scheme of iterative hybridization. STUDY DESIGN: Starting from a mixture of random oligonucleotides, through several rounds of hybridization with 39 type-specific GP5+/6+ L1 sequences, we aimed to obtain specific probes to discriminate between these HPV types. RESULTS: In vitro selection led to pools of specific probes, from which individual probes were cloned and tested for their diagnostic performance at ambient temperature. Typically, 10-fold stronger hybridization signals were obtained between each of the selected probes and their specific targets compared to signals with the remaining 38 HPV types. High sensitivity and specificity of selected probes was demonstrated a series of clinical samples in the hybridization assay. CONCLUSIONS: A new panel of probes for detecting HPV types is described. Probes can be adapted for use in a simple clinical setting, or incorporated into different detection systems.

An in vitro selection scheme for oligonucleotide probes to discriminate between closely related DNA sequences.

Using an in vitro selection, we have obtained oligonucleotide probes with high discriminatory power against multiple, similar nucleic acid sequences, which is often required in diagnostic applications for simultaneous testing of such sequences. We have tested this approach, referred to as iterative hybridizations, by selecting probes against six 22-nt-long sequence variants representing human papillomavirus, (HPV). We have obtained probes that efficiently discriminate between HPV types that differ by 3-7 nt. The probes were found effective to recognize HPV sequences of the type 6, 11, 16, 18 and a pair of type 31 and 33, either when immobilized on a solid support or in a reverse configuration, as well to discriminate HPV types from the clinical samples. This methodology can be extended to generate diagnostic kits that rely on nucleic acid hybridization between closely related sequences. In this approach, instead of adjusting hybridization conditions to the intended set of probe-target pairs, we 'adjust', through in vitro selection, the probes to the conditions we have chosen. Importantly, these conditions have to be 'relaxed', allowing the formation of a variety of not fully complementary complexes from which those that efficiently recognize and discriminate intended from non-intended targets can be readily selected.