| Summary: | 碩士 === 國立交通大學 === 生物科技系所 === 94 === The need for accurate and reliable methods for animal species identification has steadily increased during past decades, particularly with the recent food safety issues and the overall crisis of biodiversity primarily resulting from the huge ongoing illegal traffic of endangered species. Recent food scares (e.g. bovine spongiform encephalopathy, avian influenza, foot-and-mouth disease, etc.), malpractices of some food producers, religious reasons, food allergies and genetically modified organisms (GMOs) have tremendously reinforced public awareness regarding the composition of food products. A relatively new biotechnological field, known as species molecular identification, based on the amplification and analysis of DNA, offers promising solutions. This DNA-base method used is based on the presence of species-specific sequences of DNA in meat and the possibility of detecting such sequences specifically. In particular, the mitochondrial cytochrome b (Cyt b) gene sequence has proven helpful for this purpose. Numerous studies showed that Cyt b is adequate for the discrimination of different species of poultry and livestock with cluster analysis. With the fast improvements in molecular biology and technology, alternatives to ‘classical’ DNA sequencing are being developed that present interesting perspectives for species identification studies. Therefore, microarray technology can now open up new perspectives for biodiversity monitoring.
In the present study, we showed using primer number as possibly minimal to simultaneously amplify and quantify specific mitochondrial DNA (mtDNA) fragments of animal source in whole blood or skeleton muscle. These primers were designed according the alignment results of identifying highly homologous regions within the Cyt b gene by bioinformation software and each primer contained a maximum of three mismatches to all animal species wherever possible. The universal primer sets 19 - 23 mer in length was designed manually on an alignment including all Cyt b sequences. In this approach, 5 universal PCR primer pairs in 3 groups of universal primer sets (named PAL primer sets) generating a short PCR fragment (SPF) were designed for amplification of the DNA fragment of mitochondrial Cyt b ranging from 103 bp to 116 bp. The PAL primer sets permit ultrasensitive detection in 57 animal species, including livestock, poultry and relevant species. To identify specific animals by hybridization, 57 specific probes in size of 50 mer inter-primer sequences were deduced from the alignment of SPF sequences. These 50 mer probes modified with aminolinker at the 5’-end were synthesized and immobilized onto a chip slide glass to create the mtDNA Chip. Each matrix contained 57 specific-species oligonucleotide probes, two control probes, and one blank in one chip was duplicated. The specific-species Cyt b fragments were amplified by universal primer sets modified with fluorescent Cy3 or Cy5 at 5’-end. The fluorescent labeled-targets used to hybridize the mtDNA Chip to generate designed profiling for species identification. Species identification and quantification were tested from the mixtures of different animal species by the multiplex PCR and fluorescent hybridization of mtDNA Chip.
In this study, reference animal species were obtained from 13 different vertebrate animals (C. moschata, G. gallu, S. camelu, A. cygnoides, M. gallopavo, C. familiaris, F. catus, M. musculus, O. cuniculus, B. taurus, R. norvegicus, S. scrof, and O. aries) from commercial sources. The SPF were amplified using the PAL universal primer set A-f, B-f, and AB-r for C. moschata, G. gallus, S. camelus, A. cygnoides and M. gallopavo. F. catus, C. familiaris, M. musculus and O. cuniculus used the PAL universal primer set C-f and C-r. R. norvegicus, B. taurus, S. scrofa and O. aries used the PAL universal primer set D-f, E-f, and DE-r. The discrimination of 13 animal species was based on PCR or multiplex PCR amplification of target regions of mtDNA Cyt b genes using PAL primer sets, followed by analysis of the amplified DNA by hybridization with the mtDNA Chip. In the results, it was clear that the multiplex PCR technique can detect specific mitochondrial Cyt b gene fragment of these animal species from the mixture of either species alone or from mixed 2 - 5 animal species. These results indicate that the 50 mer oligonucleotide array containing 57 species-specific probes appears to be specific to their corresponding target genes. The limit of hybridized spots intensity on the chip had tested hybridization signals changed were observed with template DNA proportions using 1:1, 1:0.1, and 1:0.01. In this result, the hybridization signal intensity of the spots was significantly higher than the background signal when the B. taurus in 1% and S. camelus in 1%. The design of this ‘species identification’ chip was done by visually choosing probes, which were generally accurate and robust. This might be the easiest approach for a low-density chip including up to 57 species or more species.
In these studies showed that 50 mer mtDNA Chip had the discrimination power as for species identification in poultry and livestock. The mitochondrial Cyt b sequences in the mtDNA Chip would be promising markers for the species identification of mammals. It can be used more genes in combination to enhance redundancies and thus robustness of a specific chip including mammals in the future. This approach for a diagnostic microarray-chip is straightforward and simple. It represents an important idea for the application of a new method particularly in the field of ecology and agriculture. Once a chip has been designed and is available, any small laboratory should be able to carry out the diagnostics in a short time. Of broader use, and therefore economically more relevant, could be a ‘Biodiversity-Chip’, containing a large number diagnostic features to distinguish key species in the taxa of bacteria, lichen, molluscs, insects, fungi, mammals, etc.
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