Application and Evaluation of RT-PCR—ELISA for the Nucleoprotein and RT-PCR for Detection of Low-Pathogenic H5 and H7 Subtypes of Avian Influenza Virus

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Abstract
Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.