Objective To establish a specific,sensitive and applied method for the detection and differentiation of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus.Methods Based on the genomes sequence analysis,6 pairs of primers were designed.The special capture probes of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus were amplified,cloned and sequenced.Then the microwell plates were precoated using these capture probes,and the forward primers were labeled using biotin.The samples were then amplified using the biotin labeled forward primers and reward primers.The microwell plate hybridization was processed for detecting and differentiating the virus.The precoated DNA concentration,precoated time,hybridization temperature and hybridization time were optimized carefully.Results The A value of positive samples were over 0.5,while the average A value of the negative samples was less than 0.1.The S/N value exceeded 10.0.Sensitivity experiment suggested the method of PCR-ELISA could detect the virus RNA in 107 times dilution,while RT-PCR could detect the virus RNA in only 106 times dilution.The stability experiment of PCR-ELISA using DVⅠ suggested that the within-batch coefficient of variation was 6.21%,the between-batch coefficient of variation was 9.92%;the within-batch coefficient of variation in negative control was 1.92%,and the between-batch coefficient of variation in negative control was 3.68%.No visible changes were found on the performance of the coated microwell plates when stored in 4℃for 6 monthes.Conclusion PCR-ELISA is a more sensitive and specific method than RT-PCR is in the early detection and type identification of dengue Ⅰ-Ⅳ types virus,Japanese encephalitis virus and yellow fever virus.

Hepatitis C viral RNA in sera from 10 patients with post-transfusion hepatitis in Shanghai (China) were amplified By the nested PCR with primer sets deduced from the American prototype HCV RNA 5' -untranslated region. Eight of 10 cases (80%) were positive. And manipulations of PCR were simplified by combination of reverse transcription step and the first round PCR reaction. Our results indirectly proved that 5-untranslated region of HCV RNA of Shanghai showed a high degree of comservation and homology compared with isolates from some regions in the world.

Objective To screen and identify the dengue virus-specific antigens,then establish the ELISA detection method for dengue virus antibody.Methods Using bioinformatic software DNAStar and ANTHEPROT to analyze the hydrophilicity,flexibility,surface probability and antigenicity of dengue virus type 1-4,Japanese encephalitis virus and Yellow fever virus M,E and NSI protein amino acid sequence and also consider the influence of secondary structure.Then in accordance with epitopes localion and amino acid sequence similarity,forecast the share and specific epitopes.Reference the sequence information of different dengue virus strains in GenBank to analyze the epitopes conservative.Based on the results of bioinformatic analysis,5 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-C2x or pET32a.Then the vectors was transferred into E.coli Rosetta( DE3 ).lsopropyl-β-D-thiogalactoside(IPTG) was used to induce the expression of gene segments.SDS-PAGE were used to identify the expression proteins,and the antigenicity were tested using Western blot.Using the antigen selected by Western blot,ELISA method for dengue virus antibody detection was established.Results Eighty shared epitopes and 25 specific epitopes were forecasted,and 5 antigenic fragments encloude analyzed epitopes from dengue virus type 2 and 3 were expressed in E.coli successfully.One dengue virus type 1-4 shared antigens (Den-Ag5),one dengue virus type 2 and 4 shared antigens( DenAg3),one dengue virus type 1-3 shared antigens(Den-Ag2) and two dengue virus type 1,2 and 4 shared antigens( Den-Ag1,Den-Ag4)were conformed using Western blot.Using antigens Den-Ag5,Den-Ag1 and DenAg2,the ELISA method for dengue virus antibody detection were established.Conclusion Based on the bioinformatic analysis and Western blot verification,5 dengue virus specific antigen were conformed,and the ELISA detection method for dengue virus antibody were established.

OBJECTIVETo screen and identify dengue virus type 2 specific antigens and establish an enzyme-linked immunosorbent assay (ELISA) for detecting dengue virus type 2 antibody.

METHODSUsing the bioinformatic software DNAstar and ANTHEPROT, we analyzed the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus, and Yellow fever virus M and E protein amino acid sequences, and also evaluated the influence of secondary structure. The specific epitopes of dengue virus type 2 were predicted according to the epitope location and amino acid sequence similarity, and the epitope conservation was assessed using the sequence information of different dengue virus type 2 strains in GenBank. Based on the results of bioinformatic analysis, 5 specific epitopes were amplified and inserted into the prokaryotic expression vector pET32a, which were transferred into E. coli Rosetta (DE3) for expression of the proteins. SDS-PAGE and Western blotting were used to identify the expressed proteins and test their antigenicities. The antigen selected by Western blotting was used to establish the ELISA system for dengue virus type 2 antibody detection.

RESULTSBioinformatic analysis predicted 8 possible dengue virus type 2 specific epitopes, and 6 of them were efficiently expressed in E. coli. Western blotting confirmed 1 dengue virus type 2 specific antigen, the ELISA system for dengue virus antibody detection was successfully established using this specific antigen.

CONCLUSIONWe have obtained a dengue virus type 2 specific antigen and established an ELISA system for detection of dengue virus type 2 antibody.

Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Computational Biology ; Dengue Virus ; classification ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunodominant Epitopes ; Software