Optimization of universal primer set for the identification of all Influenza A viruses using real-time PCR

Avian Influenza Virus (AIV) occurs worldwide among aquatic wild and may mutate into a strain capable of efficient human-to-human transmission and cause a pandemic that could kill a large fraction of the human population. The purpose of this study is to efficiently identify and diagnose the virus for immediate treatment and prevention. Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR or qPCR) was identified as the most efficient way to detect AIV in previous laboratory research, however only one pair of primers was used. In order to further optimize the test and make it commercially available in the future, we first tested an eight pair primer set via RRT-PCR by using standard AIV isolates from Dr.Gaimbrones lab (Auburn University). Using the AIV isolates, the optimal cDNA and primer concentrations were determined, this aided in the identification of the most sensitive primer set. Primers PA, HA, NP, and NA were able to detect the cDNA of positive AIV isolates at a low concentration of 0.0039 mg/ml. AIV cDNA were detected by primers M and HA at its lowest concentration of 0.0001 and 0.01 pmol/l. In order to further test our lab results, 20 fecal samples from aquatic wild birds were collected from Alabama Shakespeare Park located in Montgomery, Alabama during the winter season. The samples were analyzed using RRT-PCR. HA primer set detected 10 positive samples for Avian Influenza and is the most sensitive primer set to be used in RRT-PCR. The field sample detection confirmed laboratory results and revealed that local aquatic birds are actively shedding low pathogenic avian influenza virus(LPAIV). The optimization of all primer sets used in this study will aid in disease surveillance, facilitate quick diagnosis of Influenza A viruses, and implement an effective prevention of flu pandemic.