An amplification-free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up-converting phosphor technology.
OBJECTIVE
Development of a generally applicable sensitive hybridization-based assay devoid of any target amplification for the detection and identification of (pathogenic) bacterial and viral species.


METHODS
Using a sandwich hybridization format, the presence of a species-specific nucleic acid sequence is detected by means of Lateral Flow (LF) and Up-converting Phosphor Technology (UPT, a luminescent tracer). As a model, detection of the pathogen Streptococcus pneumoniae was investigated using a probe against the single-copy lytA gene.


RESULTS
Detection of S. pneumoniae (in particular a 1200 p lytA sequences) required less than 1 ng of genomic DNA (approximate size 2.2 Mb). Hybridization and detection were performed in a complex background containing 10 microg fish sperm DNA.


CONCLUSIONS
The results indicate the possibility to detect nucleic acid targets in nonamplified DNA samples using easy, inexpensive, amplification-free hybridization-based assays and the ultra sensitive UPT reporters. Employment of UPT allows to by-pass target amplification and therefore brings genetic-based testing a step closer to the point-of-care environment. Detection of S. pneumoniae with only 1 ng of DNA indicates a potential for applications in the field of infectious diseases.
Zuiderwijk
Michel
M
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
Tanke
Hans J
HJ
Sam Niedbala
R
R
Corstjens
Paul L A M
PL
D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics*
D004269:DNA, Bacterial