Development of a microplate immunocapture rt-pcr assay for the detection of avian influenza virus

Abstract

Avian influenza viruses (AIV) cause infection processes in several species of birds and is particularly important in chickens, varying from unapparent to severe disease and death, depending on the host and virus strain. AIV infections can be diagnosed by analyzing tracheal or cloacal swab samples collected from infected birds, but when highly pathogenic AIV is suspected to be involved in an outbreak, in addition to oral and cloacal swabs, other tissues should be often targeted to monitor this infection in birds. The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), including the conventional and real-time RT-PCR techniques have been routinely used for the rapid detection of Matrix glycoprotein gene (M gene) of AIV and for the laboratory diagnosis of this virus infection. Despite the availability of various RNA extraction methods for using in RT-PCR, isolation and detection of viral RNA are still difficult due to the unstable nature of viral RNA molecules and the presence of PCR inhibitory substances in biological samples. In this study, a simple method using polyclonal antibodies from AIV hyperimmunized chicken sera were used for developing a microplate immune-capture (MIC) method to recover viral RNA from samples containing the H2N2 AIV strain and the results were compared to a standard commercial method recommended for AIV RNA extraction; the Ambion H MagMAXTMkit; followed by the application of conventional and real-time RT-PCR methods. The results showed that both RNA extraction methods were equally efficient in detecting the H2N2 AIV in conventional and real-time IC-RT-PCR, without generating non-specific detection of non-related avian RNA-virus pathogens, such as Newcastle disease virus, avian infectious bronchitis virus and Gumboro Disease Virus. Comparable analytical sensitivity was also found in RT-PCR techniques by testing RNA obtained form MIC or standard RNA extraction, either when AIV was tested in allantoic fluid suspensions or in spiked tracheal and cloacal swab samples. In conclusion, the MIC protocol reported in the present study can be advantageously used in extracting highquality RNA for accurate detection of AIV from tracheal and cloacal swabs, or allantoic fluid samples by conventional and real-time RT-PCR techniques and could be an alternative to the imported commercial RNA extraction kits.