| Morbillivirus belongs to Mononegavirales,Paramyxoviridae,Paramyxovirinae.According to the8thof international classification of Virology Committee meeting,there are six major viruses in morbillivirus. They are canine distemper virus,measles virus, Peste des petits ruminants virus, rinderpest virus, phocine distempervirus and dolphin morbillivirus virus. All of them may cause serious viral disease intheir respective host. Morbillivirus may cause public health danger, social impactand economic loss seriously. They had broad-spectrum Virus host, highpathogenicity and high mortality which have attracted the attention of researchers.There are some method such as ordinary RT-PCR, real-time quantitative PCR,serological and immunological means that have been applied to scan morbillivirus,but most of them are designed to test one or two viruses. There is not one processcan identify all the six virus in a route. The purpose of this study is to establish arapid, accurate and sensitive high-throughput microarray platform to detect measlesvirus, canine distemper virus, Peste des petits ruminants virus, rinderpest virus,phocine distemper virus and dolphin morbillivirus virus in one assay. Thismicroarray could be a rapid and effective diagnostic tool for clinical diagnosis,prevention and control of infectious diseases and anti-terrorism.All the six virus genome sequence were download from NCBI website,meaningful species-specific sequence of each virus were found by using biologicalsoftware clustal w which is important for oligo probe design. Probe ofspecies-specific, uniform length, similar Tm values were designed using molecularbiology software Primer Premier5.0. Samples were hybridized to microarray afterbeing amplified by fluorescent primers. Pictures were scanned by laser and datawere analyzed by Genepix4100A. Sensitivity, specificity, reproducibility andstability of microarray were proved by both cultured virus and clinical specimens.38clinical specimens with suspected disease of canine distemper were detected bygene chip of Jilin, Liaoning and Heilongjiang province.The chief results of the study were listed as following: 1. The designment of morbillivirus oligo probes on species level and theirvalidation by bioinformatics.Species-specific sequence was selected after comparing the downloadedgenome of morbillivirus. Six25mer probes were designed by Primer Premier5.0forevery virus with Tm value about48±5℃. All probes have similar hybridizationparameters. The validation of conserved sequence and species-specific probedesignation were tested by bioinformatics. At last, six species-specific oligo probeswere designed and selected.2. Multiplex PCR amplification for morbillivirusPrimers for the six major morbillivirus were designed according to the sameconserved sequence with probe. All six pare of primers were mixed to detectcultured virus, clinical specimens and synthesized virus in one assay. There werepositive signal for CDV, MV, DMV, PDV, PPRV and RpV while the signals ofcontroled Vero, CPIV and healthy dog were not detected.3. The establishment of microarray for morbillivirus species-specific detection.Microarray was prepared by MG2-610. Probes of40pmol/μl were spotted ontothe aldehyde slides at25℃and70%humidity. Before it can be used forhybridization, the chip must be disposed by NaBH4. Hybridization condition wasoptimized and microarray for species-spefic morbillivirus identification was set up.To test the species-specificity of the probes in the microarray, the target CDV,PPRV, CPIV, Vero, synthesized MV, RPV, PDV, DMV, and tissue of healthy dogwere prepared according to the method mentioned above and hybridized onto themicroarray. The probes only hybridized with their respective target viruses andnon-specific or cross-reactive signal was not observed CDV with TCID50as10-4wasdiluted by10folds and detected by microarray and PCR, and microarray can detectCDV as a minimum level as10-6TCID50which is103times higher than PCRmethord.4. Application of microarrayCDV can infect dogs, ferret, Raccoon, giant panda, cat and other animals. Itwas a great harm for domestic dogs, pets, economic animals, wild life, zoo andlaboratory animals. Microarray were used to detect22simulated unknown clinical specimens, and the detection ration of chip is100%. Clinical specimens of dog, foxand raccoon which were sent from Jilin, Liaoning and Heilongjiang province weretested for CDV by the microarray. Among the38specimens there were24samplewhich were tested as CDV positive by both microarray and PCR methord.26weredetected CDV positive by microarray only,and it is the same with virus isolation.5. Establishment of visual microarrayAccording to the above experimental, the short arm are connected to the5'endof virus species-specific probes,then probes are spotted on the glass, visualdetection chip could be long-term preservati after hydration. biotin were introducedto5'end of the PCR primers. the colloidal gold-streptavidin-avidin biotin reactionsystem are used to get specific binding and visual signal. Finally use silver stainingto amplify the signal. The detection platform are specific, high-throughput, highsensitivity.The visual microarray and the usual chip of virus are complement to eachother.The former are more suitable for the basic laboratory and clinical testingIn short, six25mer oligo probes were designed on species level formorbillivirus and the specificity of them was validated by both bioinformatics andexperiments. Then low-density micorarray was prepared using the oligo probes.PCR primers were designed within the same conservation that has been used to planprobes, and multiplex PCR assay was launched to amplify the six majormorbillivirus. Microarray for morbillivirus species-specific detection was initiated.Cell culture supernatants and clinical specimen could be detected by the microarrayto test morbillivirus rapidly. Morbillivirus could be detected on species levelreferring to clinical symptoms and epidemiological characteristics when infectiousdisease outbreaks.
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