Dengue Virus Infection Diagnostic Method

Dengue virus (DEN) causes Dengue fever (DF), Dengue hemorrhagic fever (DHF) and Dengue Shock Syndrome (DSS). Dengue fever is a febrile illness. Dengue hemorrhagic fever and Dengue Shock Syndrome often prove fatal. Dengue fever virus infection is an important public health issue in the tropics and subtropics. There are still some problems in the control and prevention of Dengue virus infections. By now, no safe and efficient vaccine has been licensed. There is an increasing need for rapid early diagnosis. This study developed diagnostic methods for Dengue infec-tions from aspects of serology and molecular biology.1. Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus.The internal controls target gene were obtained by insertion of a 180bp non-related DNA fragment into RT-PCT detection targeted gene of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus. 2. Real-Time One-Step mutiplex RT-PCR with TaqMan probe for genetyping of dengue virusEarly detection of Dengue infection, especially quantitative and genetyping diagnosis, is very important for clinical treatment of patients and prevention of disease dissemination. Real-Time RT-PCR can be a good method to meet the request.In this study, complete and partial sequences of four serotypes of Dengue virus were loaded from Genbank and multiple alignment were made by the software of Bioedit.12 set of serotype specific TaqMan probes and primer pairs were designed for the real-time genotyping assay..The detection limit of each assay can be 0.1TCID50/ML,0.01TCID50/ML,0.001TCID50/ML,0.001TCID50/ML for the dengue virus type 1-4 respectively. All the four tests are genotype specific and there are also no non-specific amplification for other viruses such as hantaviruses and measles virus.3. As a simple and sensitive method, IgM capture ELISA (MAC-ELISA) is recommended by World Health Organization in the early serological diagnosis. The rE3 proteins were expressed as insoluble proteins in the inclusion bodies as shown by SDS-PAGE. After dilution renaturation, the recombinant proteins were purified by metal chelating and weak anion-exchange chromatography, western bolt results showed that those proteins can react with patient serum specifically.The rEIII fusion protein were conjugated with HRP, then purified by the molecular sieve. rEIII-ELISA methods which use rEIII fusion protein as detection antigen. were established, Commercial panbio MAC-ELISA were used as reference test, The sensitivity, specificity of rEIII-ELISA method are 83.3%,99.5%, The agreement rates of rEIII-ELISA with the commercial panbio MAC-ELISA kit were 91.11%.In addition,5 serologic specimens from Japanese encephalitis virus infected patients,20 serologic specimens from epidemic hemorrhagic fever patients'sera and 20 serologic specimens from healthy persons were tested by self-developed MAC-ELISA methods, and all the results were negative. In conclusion, The self-developed MAC-ELISA methods could be applied in early serodiagnosis of Dengue virus infections to detect virus-specific IgM.