Development And Evaluation Of Rapid Nucleic Acid Detection Methods For Common Hemorrhagic Fever Viruses

Viral hemorrhagic fevers(VHFs)is an acute infectious disease caused by multiple types of viruses with fever and bleeding as the main symptoms.The disease is widely distributed around the world.There are currently at least 20 viral Hemorrhagic fever has been reported.The main epidemics in my country are hemorrhagic fever with renal syndrome,fever with thrombocytopenia syndrome,dengue fever,and scattered imported cases of yellow fever,Zika virus disease,and chikungunya fever.With the continuous advancement of economic globalization and the rapid development of transportation,the exchanges between various regions are also constantly deepening.Some severe VHFs such as Ebola hemorrhagic fever,Marburg hemorrhagic fever and other infectious diseases are also imported risks,my country’s epidemic prevention and control is facing huge challenges.Therefore,the establishment of an on-site emergency rapid detection method for hemorrhagic fever virus is essential for early detection of pathogens and timely epidemic prevention and control.This subject has carried out the following research for this purpose.The first part of the research is based on the traditional TaqMan fluorescent probe technology,using the domestic rapid one-step RT-PCR kit,combined with the Shanghai Liferiver Magnetic induction cycler(Mic)q PCR instrument.Established Severe fever with thrombocytopenia syndrome virus(SFTSV),dengue virus(DENV),Hantaan virus(HTNV),and Heartland virus(HLV),Chikungunya virus(CHIKV),Zika virus(ZIKV),Junin virus(JUNV),Lujo virus(LUJV),Sabia virus(Sabia virus),SABV),Zaire Ebola virus(ZEBOV),Marburg virus(MARV),Lassa virus(LASV)and Yellow fever virus(YFV)13 kinds of common hemorrhagic fever virus rapid PCR fluorescent probe nucleic acid detection methods,compared with the traditional real-time fluorescent quantitative RT-PCR method,the required time is greatly shortened,the detection can be completed within 35 minutes,and the established method is performed evaluation.The sensitivity was evaluated by using the prepared in vitro transcribed RNAs of 13 viral target genes as reference materials.The lowest detection limit calculated by each virus rapid PCR fluorescent probe nucleic acid detection method is less than 60copies/μL,with good sensitivity;at the same time,simulated negative samples prepared by nucleic acid extracted from Japanese encephalitis virus and other related viruses,as well as viral infections such as adenovirus Of the clinical samples,200 ordinary human samples were evaluated for the specificity of the rapid PCR fluorescent probe nucleic acid detection method.The results were all negative,no cross-reaction occurred,and had good specificity.Use SFTSV,DENV,HTNV infected clinical samples and corresponding high,medium and low concentration simulated positive samples to verify the rapid real-time fluorescent quantitative RT-PCR detection methods of SFTSV,DENV,HTNV,HLV,ZIKV,LUJV,SABV,ZEBOV,YFV.100%of the clinical samples infected by SFTSV,DENV,and HTNV were detected,which was consistent with the results of the traditional real-time fluorescent quantitative RT-PCR method established by our laboratory.Simulated positive sample verification can be detected,and the coefficient of variation of the CT value of the verification results of each group is less than 4%,and the detection repeatability is good.The second part is based on Recombinase Polymerase Amplification(RPA)technology,using a domestically-developed multi-enzyme constant temperature rapid kit mediated by multiple functional proteins,and a number of lines designed for the conservative region of the NP gene of SFTSV Primer probes,through screening to obtain a set of best primer probes,established an isothermal rapid-amplification detection method for the RNA of the SFTS virus.The reference material uses In vitro transcribed RNA of the SFTSV prepared in the first part to evaluate the sensitivity of isothermal rapid-amplification detection method for the RNA,and compare it with the real-time fluorescent quantitative RT-PCR detection method,and use the SFTSV infected clinical samples for preliminary clinical trials verification.The results show that the established isothermal rapid-amplification detection method for the RNA of the SFTS virus can detect 100% of 100 copies/μL in vitro transcribed RNA at least.For some templates with higher concentrations,it can even achieve a positive signal within 5 minutes.At the same time,this method has no cross-reactivity to other related viruses,and has good specificity.It can also reach 100% detection of clinical samples infected with SFTSV.In this study,based on the traditional TaqMan fluorescent probe technology and Recombinase Polymerase Amplification(RPA)technology,the rapid PCR fluorescent probe nucleic acid detection method for 13 common hemorrhagic fever viruses and isothermal rapid-amplification detection method for the RNA of the SFTSV were established respectively,and conducted a preliminary evaluation of the established method.It has certain application prospects for on-site emergency detection,and provides important technical support and new directions for the prevention and control of common hemorrhagic fever viruses.