Primary Design Of DNA-Microarray For Detection Pathogens In Mariculture Animals

Disease is a serious threat to the sustainable development of mariculture industry, so how to find an effective way to alleviate the disease have been the people's concerns. The existing detection technology of pathogen has great limitation of efficiency and systematic. It is in urgent need of some high-throughput means to improve the strategy in order to break the technical bottleneck. With the growing wealth of basis data of fish pathogen, the diagnostic technology of aquatic animal disease is inevitable trend toward high-throughput techniques.According to the Blue Lightner, the Office International Des Epizooties (OIE) and the Network of Aquaculture Centers in Asia-Pacific (NACA), and other aquatic animals diseases directory, select dozens of pathogenic micro-organisms such as viruses, fungi and protozoa as research subjects which pick mariculture Pleuronectiformes fish, Perciformes fish, shrimp and Shellfish as their main host, including 14 species of fish virus, 14 species of shrimp virus, piscirickettsia, 3 species of fungi and 8 species of protozoa. There are 27969 related gene sequences were collected in GenBank, including aquaculture animal pathogens sequence 26861, quality control system sequence 1108.First, there are pathogenic microorganisms were retrieved nucleic acid sequence in GenBank carryed out BLAST search against, Omiga Multiple Alignment software than to find a variety of pathogen nucleic acid sequences conserved region and sequence specific respectively. Then,the screening of various different sequences in accordance with the host is divided into 3 categories at the same time import AlleleID 6.0 software, in accordance with primers and probe design principles, in the same project to design the parameters such as annealing temperature is very similar to amplified fragment length of 200 ~ 500 bp primers. Then according to amplified fragment sequence, to design length of 57 bp oligonucleotide probe. Finally, put the design of the probes into Omiga software again, to determine between the probes up to a maximum of more than four consecutive identical base pairs and the location of primers in the amplified region with the primer binding sites do not. Sequence information submitted to the Shanghai Sangon Biological Engineering Technology Services Co., Ltd. to synthesis of primers and probesIn this study, 31 pairs of primers and 31 corresponding oligonucleotide probes were designed in detect fish pathogen; 37 pairs of primers and 37 corresponding oligonucleotide probes were designed in detect shrimp pathogen; 8 pairs of primers and 8 corresponding oligonucleotide probes were designed in detect Bivalve molluscs pathogen. There is also the host sequence as a template to design primers and probes, composed of quality control system used to monitor the whole process of the positive internal control; as well as by the unusual host-derived sequence as a template design primers and probes used to group quality control system into a positive external control.According to primers and probes design results, in accordance with the pathogenic microorganism infections in different hosts, using the principle of gene chip, the synthesized probes in accordance with the pre-distribution, was pointed into positively charged nylon membrane by Manual Glass Slide Arrayer Replicator, after UV cross-linked the oligonucleotide membrane-array was constructed.According to laboratory and subject project cooperation unit provided infected fish and shrimp materials, some of the primers specific validation experiments have been developed in this paper. In fish chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV, VHSV, IPNV and YAV DNA or cDNA as templates respectively. Also in shrimp chips, the specific PCR product that consistent with the experimental design were amplified by nine pairs of primers against WSSV, IHHNV, TSV DNA or cDNA as templates respectively. At the same time the inner and outer positive control in quality control system were also verified. Through the synchronization PCR and two-step RT-PCR method, Digoxigenin-labeled target genes are amplified by gene specific primers and DIG-labeled dUTP, then the amplified fragments are hybridized to the membrane-array, and measured the concentration is 10 ng/μl~40 ng/μl. In DIG labeled amplification products and membrane array hybridization experiments, verify the detection of fish pathogens membrane chip 11 oligonucleotide probe hybridization specificity, including 3 oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detected EHNV, LCDV, TRBIV, LYCIV, ISKNV, RGNNV, IHNV , VHSV, IPNV and YAV pathogen respectively shrimp pathogen detection chip 12 oligonucleotide probes, including four oligonucleotide probe in quality control system, 8 oligonucleotide probe were used to detect WSSV, IHHNV, TSV pathogen respectively. Then,the membrane-chip detected ISAV and MBV synthetic gene, as well as sampling from Hebei Tangshan infected shrimp samples, has also successfully tested fish pathogen detection chip ISAV probe and shrimp pathogen detection chip MBV and HPV probe.Finally, this article analyzes the prospects of gene chip technology in the field of aquaculture animal pathogen detection,aquaculture animal pathogen detection chips will be able to fast, high-throughput detection and diagnosis of animal diseases of mariculture, systematic and comprehensive reflection of mariculture cause animal diseases and conditions, to identify effective early-warning and prevention measures to protect the health of marine aquaculture sustainable development.