Simultaneous detection of all avian influenza viruses and differentiation of H5, H7, N1 and N2 subtypes by a microsphere based assay
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Avian influenza viruses are negative sense single stranded RNA viruses. They are deadly pathogens in poultry and rapid identification of these viruses is very important because of the pandemic threat as human adapted viruses can emerge by mutation or reassortment. A multiplex microsphere assay for the simultaneous detection of all avian influenza viruses and differentiation of H5, H7, N1 and N2 subtypes was developed in this study. Multiplex RT-PCR using biotinylated primers specific to the target followed by hybridization with specific oligonucleotide probe coated microspheres in a multiplex format were performed. Streptavidin-R-Phycoerythrin was used as the reporter and the fluorescence intensity was measured by the Bioplex machine. The assay is 97.43% specific and the diagnostic sensitivity is 102.5 -102.8 EID50 of virus. Validation of the assay was performed with 102 clinical samples. This assay can be used as a rapid, sensitive and specific diagnostic test for avian influenza viruses.