Establishment Of RPA Detection Method For Five Hemorrhagic Fever Viruses And Preparation Of Positive Pseudovirus References

Viral hemorrhagic fevers(VHFs)are a class of acute infectious diseases caused by RNA viruses,with fever and hemorrhage as the main clinical manifestations and endangering human and animal life and health.These diseases are mainly caused by the Filoviridae,Flaviviridae,Arenaviridae and Bunyaviridae viruses.Hemorrhagic fevers have a high mortality rate,and most of them lack of available vaccines and effective treatment approaches.Therefore,the establishments of rapid and simple methods,especially for the field detection,are very important for detection of hemorrhagic fever viruses,for timely screening of suspicious patients,and for controlling of the spread and epidemic of these viruses.Zaire ebolavirus(ZEBOV),Sudan ebolavirus(SEBOV),Lassa virus(LASV),Marburg virus(MARV)and Yellow fever virus(Yellow fever virus,YFV)belong to hemorrhagic fever viruses,are highly infectious and lethality,and have caused outbreaks in foreign countries.Although no spreading and prevalence of these viruses have been reported so far in our country,the risk of infecting with these viruses is increasing due to the frequently communication with foreigner countries.In order to prevent it from happening,we need to carry out more in-depth study of these viruses,most importantly,we need to establish a fast and accurate technology for detection of these viruses,and to lay the foundation for the effective blocking of the spread of the viruses.At present,the laboratory testing methods for hemorrhagic fever viruses mainly include virus isolation and identification,nucleic acid detection,antigen and antibody detection.Virus isolation is the gold standard for viral disease diagnosis,but usually requires higher laboratory facilities are normally time-consuming,and has biosecurity risks.Nucleic acid detection technology is mainly through the specific amplification of the target gene to diagnose the viruses,including common PCR and real-time fluorescence quantitative PCR.Common PCR is simple,but the sensitivity is not high enough.Real-time fluorescence quantitative PCR technology is fast and has a high sensitivity and specificity,it can observe the entire amplification process in real time,and quantitative analyze the initial template.Although PCR-based nucleic acid detection methods are simple and fast,but need expensive equipment and should be tested in the laboratory.The current detection method of antigen and antibody is mainly ELISA,although the detection method has been maturely developed,the process is still more cumbersome and time consuming compared with the nucleic acid detection technology.In this study,we established a novel nucleic acid isothermal amplification technique for the detection of above five hemorrhagic fever viruses,which is represented as recombinase polymerase amplification(RPA).The technology simulates the in-vivo DNA replication of the enzyme reaction process,which can be exponentially amplified at a constant temperature of 37°C to 42°C without degeneration,and the entire amplification process can be completed within 20 minutes.RPA technology has the advantages of simple operation,low requirement of equipment,high sensitivity and specificity.In this study,several sets of RPA primers and probes were designed and synthesized according to the conserved regions of the five viruses,after screening and optimization,the RPA primers and probes were obtained with high sensitivity and specificity.Secondly,pseudoviruses containing five genes of hemorrhagic fever viruses were prepared according to the conserved fragments as a positive reference for the RPA reaction system.Finally,the RPA reaction system was optimized and compared with the Real-time PCR method,to evaluate the amplification performance of RPA reaction system.The pseudovirus was treated as a simulated sample,and then nucleic acid was extracted for the for RPA amplification.Secondly,RNA was extracted from the clinical YFV samples stored in our laboratory,and RPA amplification was performed to verify whether the established RPA amplification technique was consistent with the real-time PCR reaction system.The results showed that the RPA detection technique could effectively amplify the RNA of these five hemorrhagic fever viruses at 37°C to 42°C,and had good specificity.For amplification sensitivity,the RPA amplification of the Yellow fever virus and the Marburg virus was the same as the real-time PCR,reaching 1.5 x 102 copies /reaction,and the RPA amplification sensitivity of the Zaire Ebola virus,the Sudan Ebola virus and the Lassa virus was 1.5 x 103 copies.In the detection of clinical samples of YFV nucleic acid,its amplification sensitivity is consistent with real-time PCR.It is shown that the RPA detection technology has a good amplification effect and can be applied to the field detection.