Development Of The Diagnostic Gene Chip For Detecting Four Cannie Respiratory Viruses

The aim of the study was to develop a PCR-based DNA microarray technology and a randomly primer PCR-based DNA microarray technology for simultaneous detection and species identification of multiplex canine respiratory viruses, namely canine distemper virus,canine parainfluenza virus,canine coronavirus virus and canine adenovirus. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of these viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. Fluorescence is the most popular technique to identify hybridization.After amplification and labeling with HEX, the PCR products were hybridized with the DNA microarrays and species identified. Fluorescence detection usually requires a laser scanner with a confocal microscope device to achieve sensitive detection.The PCR products of the four canine respiratory viruses ranged from187to384bp, and could be species identified with DNA microarrays. The detection limits were0.1copies/ul for each virus. And the test showed no cross-reaction to DNA extracted from Rabis,Peste des petits Ruminants virus and so on. Compared with the results of PCR, this multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of multiplex canine respiratory viruses.High throughput analysis of DNA in low concentration and small volume is an import.1.The designment of multiplex canine respiratory viruses oligo probes on species level and their validation by bioinformatics.Species-specific sequence was selected after comparing the downloaded genome of canine respiratory viruses. Four25mer probes were designed by Primer Premier5.0for every virus with TM value about48±5℃. All probes have similar hybridization parameters. The validation of conserved sequence and species-specific probe designation were tested by bioinformatics. At last, four species-specific oligo probes were designed and selected.2.The establishment of microarray for canine respiratory viruses species-specific detection. Multiplex PCR amplification for canine respiratory viruses.Primers for the four major canine respiratory viruses were designed according to the same conserved sequence with probe. All four pare of primers were mixed to detect cultured virus and clinical specimens virus in one assay. There were positive signal for CDV, CCV, CPIV and CAV2while the signals of controled RV, PPRV and healthy dog were not detected.Microarray was prepared by MG2-610. Probes of30μmol/μl were spotted onto the aldehyde slides at25℃and70%humidity. Before it can be used for hybridization, the chip must be disposed by NaBH4. Hybridization condition was optimized and microarray for species-spefic canine respiratory viruses identification was set up. To test the species-specificity of the probes in the microarray, the target CDV, CCV, CPIV, and CAV2were prepared according to the method mentioned above and hybridized onto the microarray. The probes only hybridized with their respective target viruses and non-specific or cross-reactive signal was not observed CDV with TCID50as10-4was diluted by10folds and detected by microarray and PCR, and microarray can detect CDV as a minimum level as0.1TCID50which is100times higher than PCR methord.3.The establishment of microarray for PCR amplification of randomly primed cDNA of canine respiratory viruses. We designed two randly primer including primer A and primer B.The difference between the two primers was that primer A had nine N at end of the secquence.The secquences of primer A and primer B were differently from other viruses. But this way was only used for RNA virus.We selected TM value about48±5℃.Check the products on a2%agarose electrophoresis gel. A smear from approximately200-2000bp should be visualized.In short, four oligo probes were designed on species level for canine respiratory viruses and the specificity of them was validated by both bioinformatics and experiments. Then low-density micorarray was prepared using the oligo probes. PCR primers were designed within the same conservation that has been used to plan probes, and multiplex PCR assay was launched to amplify the four major canine respiratory viruses. Microarray for canine respiratory viruses species-specific detection was initiated. Cell culture supernatants and clinical specimen could be detected by the microarray to test canine respiratory viruses rapidly. Canine respiratory viruses could be detected on species level referring to clinical symptoms and epidemiological characteristics when infectious disease outbreaks.