Devlepoment Of Nucleic Acid And Antibody Methods For Detection Of Lethal Ebola Virus

Ebola virus (EBOV) is a zoonotic pathogen which cause severe infectious diseases ofEbola hemorrhagic fever. The disease originated in1976Ebola River. Ebola hemorrhagic feveris one of the most acute, potent, deadly infectious diseases. Once a person infected with Ebolavirus, the mortality rate can be as high as88%. World Health Organization considered it asone of most serious hazards to human virus, it is classified as Level4virus, OIE listed it as aClass A disease. Although China has not found EBOV, but as China’s growing trade withAfrican countries, the disease will pose a potential threat to our country.In order to prevent Ebola into our country, we have established two fast and reliabletechnique for the diagnosis of Ebola virus. One is the fluorescence quantitative RT-PCRdetection methods for quantitative detection of the Ebola virus to detect the glycoprotein(GP)of th SEBOV and ZEBOV glycoprotein (GP). In order to maintain the usefulness offluorescence quantitative RT-PCR method, we chose the highly conserved region of theglycoprotein as amplification primers and probes. The standard samples of10-fold dilutionseries of pblue-SG and pblue-ZG proceed Real-time PCR amplification, made standard curveand detected repeatability, accuracy and specificity through condition optimization. The resultsshow that correlation coefficient of the Sudan and Zaire standard curve both were greater0.99;sensitivity detection were up to1.0×101copies which were higher than conventional PCRmethod; the detection of seven control fulminating disease specimens were negative. Second,We developed an indirect ELISA method for detecting Ebola virus (EBOV) antibodies based onthe recombinant protein expressed in Escherichia coli (DE3) strain.VP40is the most abundantvirion protein and the highly conservative. The recombinant plasmids pET22b-VP40transformed into E.coli BL21, and induced at1.0mmol/L IPTG and37℃. Expressed proteinwas purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Westernblot. We used the recombinant VP40as coating antigen and established indirect ELISA methodfor Ebola antibody detection, and the results showed that both sensitivity and specificity were100%.In addition, The experiment established successfully two high specificity, sensitivity andstability detection methods of Ebola virus, which laid a foundation for rapid diagnosis andepidemiological investigations after the outbreak of Ebola haemorrhagic fever.