Establishment And Application Of Multiplex Real-time RT-PCR Based On The TaqMan Probes For Detection Of Arboviruses

Arboviruses comprise a group of viruses which cause natural foci of disease and zoonosis by blood-sucking arthropads bite sensitive vertebrate.They are mainly include genus Alphavirus in Togoviridae,genus Flavivirus in Flaviridae and genus Orthobunyavirus in Bunyavirudae,such as Chikungunya virus(CHIKV),Sindbis virus(SINV),Eastern equine encephal omyelitis virus(EEEV),Western equine encephal omyelitis virus(WEEV),Ross river virus(RRV),Venzuelan Equine Encephal omyelitis virus(VEEV)in genus Alphavirus.Yellow fever virus(YFV),Dengue virus(DENV),Japanese encephalitis virus(JEV)and West Nile Virus(WNV)in genus Flavivirus.La Crosse virus(LACV),Tahyna virus(TAHV)and California encephalitis virus(CEV)in genus Orthobunyavirus.The viral transsion to human and livestock through insects causes human disease and death,it also causes disease of a large number of livestock,and even death,resulting in tremendous economic loss and had been regarded as a public health problem of every country.Arboviruses distribute widely,but mainly distributes in temperate,subtropical and tropical zones.Most arboviruses are only distributed in a region or a continent,but can be spread to different places with crowd,migration of host and vectors.Therefore,arboviruses carried by medical vectors shoud be strengthen surveillant.At present,detection of arboviruses depend on virus isolation,ELISA and/or molecular biology techniques.But these methods owned some disadvantages such as taking long time,low sensitivity,lacking of specific antibodies.Therefore,multiplex real-time RT-PCR based on the TaqMan probes specific to genus Alphavirus,Flavivirus and Orthobunyavirs were developed and applied for the detection of arboviruses carried by mosquitoes collected in Yunnan frontier regions.Objective:To develop a multiplex Real-time RT-PCR method for detection of arboviruses,especially the arboviruses carried by immigrate medical vectors.Methods:The complete genome sequences of strains of genus Alphavirus,Flavivirus and Orthobunyavirus were searched from GenBank.The conserved sequences of the viruses were aligned and blasted with DNAstar software.Then several pairs of primers and probes were designed with the Beacon Designer 7.0.RNA of GETV,JEV(strain MB090509)and TAHV(strain XJ0625)were used as positive controls.Mosquitoes collected in Yunnan frontier regions were detected by the developed method.Results:TaqMan Real-time RT-PCR for genus Alphavirus,Flavivirus and Orthobunyavirus,respectively,had good sensitivity and specificity.The low detecting limit of GETV was 2.9×103copies/μL,CHIKV was 1.74×103 copies/μL.JEV was 1.3×103 copies/μL,YFV was 6.6×102copies/μL,DENV(serotype 1-4)was 9.3×102 copies/μL-1.46x103 copies/μL,TAHV was 5.76×102copies/μL.There were no cross-reaction between Alphavirus,Flavivirus,Bunyavirus and Banna virus in singlet system.Multiplex TaqMan Real-time RT-PCR for genus Alphavirus and Flavivirus,and Multiplex TaqMan Real-time RT-PCR for genus Alphavirus,Flavivirus and Orthobunyavirus also had good sensitivity and specificity.The low detecting limit results of the multiplex TaqMan Real-time RT-PCR for genus Alphavirus and Flavivirus and for genus Alphavirus,Flavivirus and Orthobunyavirus were the same as the TaqMan Real-time RT-PCR of the singlet system.8 pools were detected as positive of the 201 pools of mosquitoes by using the developed multiplex real-time RT-PCR method,and 8 strains of JEV were obtained by traditional virus isolation.Conclusion:Multiplex TaqMan Real-time RT-PCR method for the detection of genus Alphavirus,Flavivirus and Orthobunyavirus,is broadly worthy in arboviruses detection.