조류 검역을 위한 DNA 마이크로어레이의 개발

Abstract

Owing to the rise of global trade volume and the movement of biological resources between every country, the importance of animal and plant quarantine is increasing in this decade. In the rapid and accurate quarantine system, it is necessary to prevent a domestic economic loss of agri-food industries and an influx of virus and bacteria which causes diseases to human, animal and plant. Influenza A viruses like an avian influenza can mutate some sequences of their HA and NA genes, by means of an antigenic drift, and also create new subtype via antigenic shift called reassortment. Although various methods of molecular genetics such as RT-PCR, DNA sequencing and RFLP have been commonly used for animal and plant quarantine, new diagnostic tool was need for more simple, fast and accurate genetic analysis. Microarray based technology provide a cost effective, simple and rapid method not only to discriminate animal species but also to diagnose virus. First, we developed a DNA microarray for discrimination of 17 Korean bird species through analysis of mitochondrial Cytochrome oxidase I (COI) which is mainly used for DNA barcode of animal. In order to select and design of optimal probes from 149 COI gene sequences of 39 avian species, in-house software was developed. This microarray can be applied for biodiversity monitoring, epidemiological studies of avian influenza, analysis of bird strike on aircraft. Second, a diagnostic microarray was developed to detect potential pandemic AIV subtypes H5, H7, and H9 and discriminate between high pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). Universal primer for amplification of HA gene were designed and then used for amplification of 46 viral RNA and 34 unknown RNA samples. We analyzed 392 HA gene sequences from avian disease lab in Konkuk University and influenza virus resource database of Genebank. 87 candidate probes were selected and designed according to probe length, Tm of probes and position of mutation in the probes. Among 34 unknown samples of avian influenza, H5, H7 and H9 subtype were identified by our microarray and these results were consistent with RRT-PCR and DNA sequencing. In this study, it is time consuming and inefficient to select candidate probes in analyzing hundreds of DNA sequences as a manual work, we made in-house software and a web-based program to design optimal probes. We developed a diagnostic microarray for avian species identification and AI virus subtyping and pathotyping. Our result demonstrated that oligonucleotide microarray technology provides a reliable, cost effective and rapid method for identification of avian species and detection of avian influenza virus.