Developments And Applications Of West Nile Virus Rapid Diagnosis Methods

West Nile virus(WNV) infection is an acute febrile, arthropod zoonosis, which can lead to fever, erythra, lymphadenectasis and encephalitis caused by WNV. With the frequent outbreak in humans and animals, it lead to a large number of casualties and considerable economic losses. As a result, WNV infection has become a global concern, and it has been listed as one of the major global epidemic by World Health Organization(WHO) and World Organization for Animal Health(OIE).Current researches indicated that there is a presence of WNV antibody in birds in China, South Korea, Japan and other Japanese encephalitis virus endemic countries. However, with the appearance of WNV in our neighboring countries, the appearance of positive serum in Xinjiang, and the increasing international exchanges, the risk of imported WNV infection into China has greatly increased, resulting in our enhanced vigilance. So a rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Research target:In order to provide simple, rapid and sensitive detection technology and method for the epidemiological investigation, rapid diagnosis, monitoring and immune effect monitoring of WNV infection, this study developed research for nucleic acid and antibody detection of WNV, three detection methods of WNV infection were developed, namely indirect ELISA for detecting West Nile virus antibody, real-time RT-PCR for West Nile virus nucleic acid detection and visual detection of West Nile virus nucleic acid. Research contents:1. Development and application of indirect ELISA for detecting West Nile virus antibody;2. Development and evaluation of one step real-time RT-PCR for West Nile virus nucleic acid detection;3. Development and evaluation of visual detection for West Nile virus nucleic acid combined with a vertical flow visualization strip. Research method:1. Development of indirect ELISA for detecting West Nile virus antibodyConstructing a recombinant plasmid pG-EDIII, and transforming into E.coli strain DE3 for prokaryotic expression of the target protein. Identifying the protein by SDS-GAGE and Western blot to verify its molecular weight and reactivity. After purification, dialysis and concentration, the West Nile virus EDIII protein was stored at-20℃.Establishing an indirect ELISA which can detect WNV antibody by the usage of the purified WNV E protein domain III as coating antigen. After the reaction conditions were optimized, the specificity, sensitivity, repeatability of the indirect ELISA was analyzed to evaluate practicality of the method.2. Development of one step real-time RT-PCR for West Nile virus nucleic acid detectionSelecting conserved sequence after analysis of strains of WNV isolated over various years from different regions and disparate species, then a pair of primers and specific TaqMan probe were designed and synthesized. Through the optimization, the 10-fold serial diluted RNA template was amplified by real-time RT-PCR and standard curve was drawn. And then, the real-time RT-PCR for West Nile virus nucleic acid detection was developed and optimized. The specificity, sensitivity, reproducibility and clinical samples were evaluated to analyze practicality of the method.3. Evaluation of the visual detection for West Nile virus nucleic acid.Selecting conserved sequence after analysis of 30 strains of WNV isolated over various years from different regions and disparate species, then Six primers, targeting 8 distinct regions of the gene were designed and synthesized. Through optimization of reaction conditions and evaluation of amplification efficiency, the visual detection for West Nile virus nucleic acid was developed. Combining this assay with a vertical flow visualization strip and carrying out the specificity, sensitivity and clinical samples evaluation to analyze practicality of the method. Research results:1. The recombinant plasmid pG-EDIII was constructed, which express WNV EDIII protein. The target protein mainly express in the form of inclusion bodies after transformed into E.coli. The target protein was purified by His Ni-NTA affinity chromatography column. An indirect ELISA which can detect WNV antibody was established using the purified WNV E protein domain III as coating antigen. After evaluation, this method has the characteristics of high specificity, high sensitivity and good repeatability, moreover, the detection results had high coincidence rate with commercial kit.2. An method of one step real-time RT-PCR for West Nile virus nucleic acid detection was developed. The optimized method has advantages of high specificity, small batch variation coefficient, good accuracy and repeatability, moreover, it has 10 times higher than the regular PCR.3. A visual detection for West Nile virus nucleic acid combined with a vertical flow visualization strip was developed. This detection method has high specificity, high sensitivity, and there is no interference of non-specific amplification in clinical. As a result, this method is suitable for clinical application.