Development Of Diagnostic Gene Chip For Detection Six Kinds Of Animal Original Zoonosis Viruses

In recent years,the occurrence of zoonosis was increasing worldwidely.The breaking out of new zoonosises and reemerging of some old ones are threatening and challenging the whole world.The governments of every country and the relevant international organizations have paid great attention to this severe public health problem.Pathogens of zoonosis including some new emerged viruses(such as SARS-CoV,avian influenza viruses,hepatitis E virus,nipah virus and rift valley fever virus),some known viruses(such as foot and mouth disease virus,vesicular stomatitis virus,rabies virus) and other potential pathogenic factors have already appeared in our country or widely spreaded in adjacent areas. The fact that those zoonosises could cause diseases or even death in animals and human reminded us enhancing the prevention and cure of animal diseases,especially controling and eliminating pathogens of zoonosis was an urgent task.Rift valley fever virus(RVFV),nipah virus(NiV),foot and mouth disease virus(FMDV),vesicular stomatitis virus(VSV),hepatitis E virus(HEV) and rabies virus(RV) were six viruses which widespreaded recently and could cause serious zoonosis.Some of susceptible animals of those viruses were same,which hindered the prevention and control of the diseases caused by those viruses.The purpose of this study was to prepare a cDNA diagnostic chip for simultaneously detection of RVFV,NiV, FMDV,VSV,HEV and RV and establish a rapid diagnosis method for the diagnosis and monitoring of those pathogens.According to the complete gene sequences of RVFV,NIV,FMDV,VSV,RV and HEV published on GenBank,highly conserved gene sequences of those viruses were located by multiple sequence alignment analysis of DNAman and Bioedit biological softwares respectively.Then,specific probes targeting those highly conserved gene regions were designed.To solve the problem of multiple serotypes or genotypes for some of the viruses.such as FMDV,HEV and VSV,three probes for each virus were designed and each probe was spotted six times to raise the capacity for virus detection.Five recombinant plasmids named pET-32a-RVFV-ORF₂.pGEM-T-NiV-P,pGEM-T-RV-N, pGEM-T-VSV-N and pGEM-T-FMDV-3ABCD respectively were constructed.Total eight recombinant plasmids including above five plasmids and the other three plasmids named pET-32a-HEV-ORF₂. pGEM-T-action and pGEM-T-wheat respectively were used as templates for PCR amplification.The products were spotted on aldehyde-coated slide as detection probes after purified by gel extraction. Totally,20 probes including 18 detection probes,one positive control probe and one negative control probe were obtained.Viral RNAs were extracted and reverse transcriped with random primers.Then the target gene conjuncted fluorescent dyes were obtained by multiplex PCR method.After optimization,four multi-PCR systems were chosen for further studies:ZH1(RVFV3,NIV3,FMDV3,RV3,VSV3),ZH2 (RVFV1,HEV2,FMDV1,NIV2),ZH3(FMDV2,RV2,HEV1,RVFV2,VSV2) and ZH4(HEV3,NIV1, RV1,VSV1,ACTIN).Those PCR reaction systems were all 50μl 0.5μl 100×aa-dUTP mixture was added to each reaction system,and PCR products were labeled with Cy5 fluorescent dyes by indirect labeling method.After optimization,the working conditions were designated as follows.The concentration of spotting solution was 350ng/μL;the hydration temperature was 45℃;the hydration time was 2 h and the formamide terminal concentration was 40%.The sensitivity experiment showed that the detection microarray system could detect as low as 1.69×10⁴,3.32×10³,1.57×10³,1.25×10³,1.51×10⁴,and 1.16×10³ copies of template RNA for RVFV,NIV,FMDV,VSV,RV and HEV respectively.The established method was applied to detect 11 samples of RV and 7 samples of HEV.The results presented positive,which were consistent with the results of RT-PCR completely.Five unknown serum samples were tested using this chip detection method,the results showed that four of them were HEV positive. The detection rate was 80%,and the results were also consistent with conventional RT-PCR.Finally,a rapid,specific,sensitive and stable gene chip method for general detection of six zoonosis viruses is established in this study.This method can be used for the diagnosis and monitoring of the six zoonosis viruses mentioned above.