Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: A rapid, sensitive DNA test to identify human papillomavirus type 16 infection

Background. A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPT(TM)) was applied to DNA (hybridization) assays fo r the detection of specific nucleic acid sequences, thereby aiming to perfo rm the test outside well-equipped laboratories. The methodology reported he re is sensitive and provides a rapid alternative for more elaborate gel ele ctrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a def ined series of cervical carcinomas. Methods: A LF assay was used to capture haptenized DNA molecules and hybrid s, which were immunolabeled (before LF) with 400-nm UPT particles. These pa rticles emit visible light after excitation with infrared in a process call ed up-conversion. Because up-conversion occurs in only the phosphor lattice , autofluorescence of other assay components is virtually nonexistent. Results: The use of the UPT reporter in LF-DNA tests, as compared with coll oidal gold, improved the detection limit at least 100-fold. UPT LF-DNA test s were successfully applied to detect (in a blind test) the presence of HPV 16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas. Conclusions: The use of UPT in LF assays to detect specific nucleic acids p rovides low attamole-range sensitivity. Hybridization and consecutive detec tion of PCR-amplified HPV16 sequences were successful in a background of 10 mug of fish-sperm DNA. The sensitivity of UPT detection in these complex m ixtures indicates that detection of viral infections without PCR or other a mplification technique is achievable. (C) 2001 American Association for Cli nical Chemistry.