A One-Step RT-PCR Array for Detection and Differentiation of Zoonotic Influenza Viruses H5N1, H9N2, and H1N1

Abstract

Rapid and comprehensive pathogen identification is crucial in zoonotic influenza diagnosis. By optimizing the design of primers and probes and reverse-transcriptase polymerase chain reaction (RT-PCR) conditions, we achieved simultaneous detection of multiple influenza and zoonotic influenza viruses, including H1N1, H5N1, and H9N2 strains, in a one-step, quantitative real-time RT-PCR array (rRT-PCR array) of RNA from multiple influenza strains utilizing a single set of conditions for RT-PCR amplification. The target sequences from all targeted zoonotic influenza viruses were cloned into recombinant RNA virus particles, which were used to evaluate sensitivity, specificity, and reproducibility of the zoonotic influenza viruses RT-PCR array. The detection limit of the array was shown to be between 10(0) and 10(1) copies per reaction, and the standard curve demonstrated a linear range from 10 to 10(6) copies. Thus, the analytical sensitivity of this zoonotic influenza viruses RT-PCR array is 10-100 times higher than conventional RT-PCR. Specificity of the one-step zoonotic influenza viruses RT-PCR array was verified by comparison of results obtained with retroviral-like particles (RVPs), which contained RNA from isolates of seasonal influenza viruses, zoonotic influenza viruses, and other pathogens known to cause acute respiratory disease. The high sensitivity, rapidity, reproducibility, and specificity of this zoonotic influenza viruses rRT-PCR array has been verified as being sufficient to detect the presence of multiple zoonotic influenza viruses in a single assay. The zoonotic influenza viruses RT-PCR array might provide rapid identification of emergent zoonotic influenza viruses strains during influenza outbreaks and disease surveillance initiatives.