Study On Detection Of Rift Valley Fever Virus Nucleic Acid And Antibody

Rift Valley Fever(RVF),an acute zoonotic infectious disease,is caused by Rift Valley Fever virus(RVFV)and transmitted by mosquito bites,direct contac t and aerosol.It mainly infects ruminants such as sheep,goats and cattle.It can cause mortality in pregnant female animals as high as 80% to 100%.The mortality rate of adult ruminants is 20%,and the mortality rate of unborn and young animals is nearly 100%.It can also cause human infection.Since the large-scale outbreak in Kenya in 1931,the disease has been prevalent in eastern,southern Africa and the Arabian Peninsula,causing enormous economic losses and public health hazards.Therefore,the Office International Des Epizooties(OIE)lists RVF as a legally reported infectious disease,which is considered as first class disease by the Ministry of Agriculture in China.So far,there are no approved commercial RVF vaccines for humans and effective the rapeutic drugs in domestic and abroad.At present,with the rapid development of economic globalization,the import and export of animal products trade are becoming more and more frequent.In addition,China is geographically close to the Arabian Peninsula so that the risk of natural infections or imported cases of foreign animals are also increasing.In July 2016,the first case of imported Rift Valley Fever diagnosed in Beijing,China,sounded the alarm for us.Therefore,it is of great significance to establish a rapid,sensitive and specific diagnostic method for RVF,so that we can achieve early prevention and control of epidemics.RVFV is a negative-sense single-stranded RNA virus,a member of genus Phlebovirus from Bunyaviridae family,has only one serotype.The genome consists of three segments: S,M,and L,of which the S fragment is 1690 nt in length.The two-way coding strategy is used.The translation strand encodes the nucleocapsid protein N,and the sense strand encodes a non-structural protein NSs.The M fragment is 3885 nt long,encoding four proteins,two envelope glycoproteins Gn and Gc,two non-structural proteins Nsm1 and Nsm2;the L fragment is 6404 nt long and encodes an RNA-dependent RNA polymerase.This paper establishes a rapid detection method for RVFV,provides simple,rapid and sensitive detection techniques and methods for rapid diagnosis,monitoring and early warning and epidemiological investigation of RVF.1.Establishment and evaluation of RVFV reverse transcription loop-mediated isothermal amplification nucleic acid visualization detection methodIn this study,a total of 25 RVFV S genes of different ages,different species and different geographical sources were sequenced,The highly conserved regions of the screening sequences were analyzed by MEGA 7 software,and 6 specific primers were designed for the conserved regions,using for Loop-mediated isothermal amplification(LAMP).By optimizing amplification time,temperature and primer concentration to improve amplification effi ciency,a method for detecting RVFV reverse transcription loop-mediated isothermal amplification nucleic acid visualization(RT-LAMP-VF)was established.And carry out specific evaluation,sensitivity evaluation,comparative evaluation with real-time PCR and preliminary evaluation of clinical samples.The results showed that the RVFV RT-LAMP-VF assay was successfully established by using artificially synthesized RNA as a template.The virus RNA of 1.94×105 copies/?L can be detected by reacting at 63°C for 20 min.After reacting for 60 min,the detection limit is up to 1.94×100 copies/?L of RNA.Using RNA,extracted from the inactivated virus of RVFV-BJ01 as a template,1.83×103 copies/?L of viral RNA was detected.In addition,this method's test results is positive for the simulated clinical sample.In summary,the method observes the amplification result by using a disposable nucleic acid visual inspection device,and realizes a visually visible and more intuitive reading while avoiding the aerosol pollution problem in the conventional isothermal amplification,and the result is accurate,sensitive,high virus specificity,no need for expensive equipment,and suitable for on-site testing.2.Establishment and evaluation of RVFV indirect hemagglutination antib ody detection methodIn this study,the ectodomain gene of RVFV Gn protein was amplified by PCR,and inserted into the prokaryotic expression vector p ET-30a(+)to construct the recombinant plasmid p ET-30a(+)-RVFV-e Gn,and the recombinant plasmid was transformed into BL21/DE3 competent state,protein expression inducing by IPTG,renatured by urea and then purified by His-tag protein purification column.It is used as an antigen to sensitize the red blood cells after hydroformylation.The RVFV indirect hemagglutination antibody detection method was established by optimizing the sensitization time,amount of antigen and reaction temperature,then the repeatability,RVFV serum samples and specificity were evaluated.The results showed that the 1287 bp target fragment was specifically amplified by PCR,and the recombinant plasmid DE3-p ET30a(+)-RVFV-e Gn was successfully constructed.The recombinant plasmid was transformed into BL21 competent cells,and the target protein was efficiently expressed.The optimal expression conditions were 30°C,using 0.8 mmol/L IPTG induction for 5 h,and the target protein was expressed in inclusion bodies.After purification,the purity of the target protein was 94.136%.Using it as an antigen,the optimal sensitization condition was determined to be 37°C,and the antigen amount of 200 ?g/m L was sensitized for 3 h.In summary,this study successfully established the RVFV indirect hemagglutination antibody detection method,which is safe,easy to operate and can be used for batch sample detection,which provides a simple and fast detection method for the evaluation of RVF new vaccine and disease monitoring.