Avian Sexing and the Detection of Newcastle Disease and Avian Influenza Viruses Using Oligonucleotide Microarrays
Date Issued
2008
Date
2008
Author(s)
Wang, Lih-Chiann
Abstract
There are numbers of wild birds rescued or quarantined in Taiwan each year. Most of them are endangered species. Their rarity poses great responsibility of us on breeding. Information of an individual bird’s sex, thus, is critical to the breeding strategies and conservation programs. Wild birds are important carriers for many pathogens, e.g. Newcastle disease virus, avian influenza virus and others. Early detection of these pathogens from carrier birds is important for the disease prevention from spreading to susceptible herds. DNA microarray, also called DNA chip, is a newly developed technique this decade. It is divided into cDNA microarray and oligonucleotide microarray based on the size of probes. Lots of molecular information is obtained at the same time through hybridization between target DNA and probes on the microarray. It provides a rapid approach to the gene analysis and pathogen detection. The purpose of this study is to develop an oligonucleotide microarray system which can precisely determine the gender of diversified avian species. Simultaneous detection and differentiation of the most two important zoonotic viruses, Newcastle disease virus and avian influenza virus, is also the main objective of this research. Most birds are vulnerable to stress. How to decrease injuries while handling, therefore, is a major consideration. Sex-related gene markers of various avian species were sequenced. The sequencing nucleotides, including the sequences retrieved from the GenBank, were aligned and analyzed, and specific primers and probes were designed. Each respective PCR method was developed, and then gradually integrated into multiplex PCR. The probes were spotted to specific positions on the microarray polymer substrate. Hybridization between target DNA and probes was performed on the array surface. The information of the gender and the carried pathogens were then obtained according to the hybridization pattern on the microarrays. We found that a cut feather including the scapus, instead of a plucked feather, is a good source of genomic DNA for bird sexing. It means this cutting method could substantially decrease injury to the examined birds. The nine designed probes on microarrays combined with two-CHD1 intron approach have successfully identified the sex of 80 avian species covering 26 families. Moreover, the 12 designed probes on microarrays targeting fusion protein gene of NDV, matrix protein gene of AIV and haemagglutinin gene of AIV also have successfully detected and differentiated different types of viruses simultaneously. Oligonucleotide microarrays, therefore, may provide potential for rapid sexing and zoonosis detection. It would be conducive to the conservation and zoonosis prevention of wild birds.
Subjects
Zoonosis
DNA microarray
Oligonucleotide microarray
Sex identification
Avian influenza virus
Newcastle disease virus
Multiplex PCR
Type
thesis
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