Rapid Subtyping and Pathotyping of Avian Influenza Virus using Chip-based RT-PCR

Frequent outbreaks of avian influenza viruses (AIV), which are influenza A viruses, result in heavy economic and national damages. However, current diagnostic methods require about 5–7 days for AIV to be confirmed, which allows enough time for the widespread dissemination of the virus. Therefore, there is a need for rapid and accurate diagnosis of AIV. Here, we developed a diagnostic method using chip-based quantitative reverse transcription-polymerase chain reaction (RT-qPCR) for the rapid identification of the major subtypes H5, H7, and H9 of AIV that influence the pathogenicity of the virus. Specific primer pairs and probes were designed to target the highly conserved Matrix (M) gene regions in influenza A viruses. Specific primer pairs and probes for subtyping were developed by targeting the conserved sequences in the haemagglutinin (HA) gene specific to each subtype. For pathotyping, specific primer pairs and probes for the highly pathogenic avian influenza viruses (HPAIV) were generated by targeting the HA cleavage site, which is highly related to the pathogenicity of the virus. Using the primer pairs and probe sets, we synthesized the major sequence of each type and constructed a standard curve to confirm their detection limit. In addition, we used a chip-based RT-qPCR method to test the samples collected from the faeces of wild birds, cloacal and oropharyngeal swab samples of infected chicken, and allantoic fluids samples incubated with inoculated eggs. As a result, AIV was accurately detected in all samples. Our study shows that by using a single DNA chip in a portable chip-based RT-qPCR device and specific primer pairs and probes, rapid and accurate AIV detection and subtyping and pathotyping is possible, which will reduce the spread of the virus, thereby reducing the economic and national damages.