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) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas.


METHODS
A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called 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 colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 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 provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 microg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.
Corstjens
P
P
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Corstjens@lumc.nl
Zuiderwijk
M
M
Brink
A
A
Li
S
S
Feindt
H
H
Niedbala
R S
RS
Tanke
H
H
D000911:Antibodies, Monoclonal; D016477:Artifacts; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics* / Q000276:immunology; D004871:Erbium; D005260:Female; D005453:Fluorescence; D006241:Haptens; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D027383:Papillomaviridae / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis*; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D015018:Ytterbium
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D006241:Haptens; D004871:Erbium; D015018:Ytterbium