Development Of Diagnostic Gene Chip For Detection Ten Kinds Of Encephalitis Viruses

Arbovirus is a group of virus which can propagate in sensitive blood-sucking insects, such as mosquito, tick, midges, and so on. However, it dosen't make its host insects pathogenic. Arbovirus could be spead by blood-sucking insects bites and induces a series of diseases called zoonotic viral disease. More than 540 Arboviruses involved in 14 virus families such as Flaviviride, Togaviride and Bunyaviridae are found all around the world. 9 viruses of them are isolated in China and it has been proved that Japanese encephalitis virus, Tick-borne encephalitis virus, Dengue virus and Sinkiang hemorrhagic fever virus are prevalent in China. Meanwhile, Japanese encephalitis virus and Tick-borne encephalitis virus are included on the list of reported communicable diseases. With the international exchanges become more frequent, the risk of arbovirus disease overseas spreading to China is growing. Therefore, the rapid and accurate method for arbovirus detection is of great significance.Gene chip is a new technology which has been developed since 1990s with the characteristics of high-throughput, parallelism, automatization, etc. In this paper, Japanese encephalitis virus(JEV), Tick-borne encephalitis virus(TBEV), Dengue virus(DENV), Rabies virus(RV), West Nile virus(WNV), Eastern equine encephalitis virus(EEEV), Western equine encephalitis virus(WEEV), Chikungunya virus(CHIKV), Rift valley ferver virus(RVFV), Nipah virus(NiV) were collected to be studied. All of the other 9 zoonosis viruses belong to Arbovirus except RV. The purpose of this study was to prepare a gene chip for simultaneously detection of JEV, TBEV, DENV, WNV, RV, WEEV, EEEV, CHIKV, RVFV and NiV and establish a rapid diagnosis method for the diagnosis and monitoring of those pathogens.According to the complete gene sequences of JEV, TBEV, DENV, WNV, RV, WEEV, EEEV, CHIKV, RVFV and NiV published on GenBank, highly conserved gene sequences of those viruses were confirmed by multiple sequence alignment analysis of DNAman and DNAstar biological softwares respectively. Then specific oligo-probes for those highly conserved regions were designed among which one probe or three probes were designed for each virus. All oligo-probes were 56-mer in length with TM between 82℃and 88℃. The homology between each oligo-probe and the other viruses'genome is less than 50%. According to the highly conserved sequences of those viruses, 15 paris of specific primers were designed by using Oligo 6.0 and Primer premier 5.0 biological softwares respectively. The synthetic oligo-probes concentration was diluted to 30μM with terminal concentration of 50% DMSO and degenerated under the condition of high temperature. Then the probes were spotted on aldehyde-coated slide in order to prepare the gene chips.Viral RNAs were extracted and reverse transcriped with random primers. After optimization, four multi-PCR systems were chosen for further studies: group A (P3, P9, P10, P12); group B (P2, P11, P13); group C (P4, P6, P8, P15); group D (P2, P5, P7, P14). The volumes of those PCR reaction systems were all 50 l. The concentration of aa-dNTPs was 20μM in each reaction systems. Then the PCR products conjuncted aa-dNTPs were labeled with Cy5 fluorescent dyes by indirect labeling method.After optimization, the hybridization conditiongs were chosen as follows. The concentration of spotting solution was 30μM; the hybridization temperature was 45℃; the hybridization time was 2 h and the formamide terminal concentration hybridization solution was 40%. Then the availability, repeatability, specificity, and sensitivity of the optimized assay were conducted. Obvious positive hybridization signals were obtained when using the 10 viruses as samples, which confirmed the availability of this chip. Two pieces of gene chips were randomly choosed from three lots at different times to verify the repeatability, which showed the best results. The specificity of the optimized assay was evaluated using YFV, FMDV, EV71 and H1N1, resulting in no specimens yielding a strong signal. The result of sensitivity test showed that the limited detection for TBEV is 87 TCID₅₀ and for JEV and RV is 9.05×10²,1.13×10³ respectively.To validate its actual testing effect, simulation clinical specimens were made using the mixture of cell supernatant infected TBEV, JEV and DENV and the brain of Balb/c mouse. The result revealed that the established gene chip inspection could detect clinical simulation specimens specifically. In a word, the gene chip method was developed to detect the 10 kinds of viral encephalitis viruses above, indicating a suitable method for rapid, specific and sensitive detection of those viruses.