Abstract
Avian influenza is a highly contagious disease, and different subtypes of avian influenza virus (AIV) have different levels of pathogenicity. A microsphere-based fluorescent assay was initially established for subtyping AIV. DNA fragments were amplified with biotinylated primers. AIV subtype-specific DNA probes with an amino-linker at the 5' end were covalently bound with carboxy-modified encoded beads. The modified beads and the denatured DNA fragments were mixed together for hybridization. Then, quantum dots-streptavidin (QDs-streptavidin) was added to conjugated biotinylated PCR products. The reaction products were screened by flow cytometry. AIV strains (such as H5N1 and H9N2) could be determined and subtyped according to their combination of encoded beads and fluorescent QDs. The method's combined sensitivity of the nucleic acids of H5N1 and H9N2 avian influenza virus at a threshold of 74 pg and 1 pg could be detected. This is a powerful method for detecting many pathogens or many types of a pathogen simultaneously.
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