Establishment And Application Of Getah Virus TaqMan RT-qPCR Detection Method

Background and Objective:Getah virus(GETV)is an arbovirus,belonging to the genus Alphavirus within the family Togoviridae.GETV is mainly distributed in Southeast Asia,Australia and other places,and it is also widely distributed in China.It infects pigs,horses and other animals through mosquito bites,which can cause fever,rash,lymphadenectasis and other symptoms.In severe cases,it can cause abortion,dead fetus and abnormal fetus.There is currently no specific treatment.In addition,antibodies to Getah virus can also be detected in human serum.In view of the widespread distribution of GETV in our country,the great harm to the animal husbandry and the potential pathogenicity to humans,there is an urgent need to strengthen the molecular epidemiological monitoring of GETV in my country to improve disease prevention and control capabilities.This study aims to establish a sensitive and specific real-time fluorescent quantitative RT-PCR method for rapid detection of GETV.Methods:We performed multiple sequence alignments on all 44 GETV genome sequences in the Gen Bank database,designed primers and probes for the highly conserved regions,and performed NCBI blast to verify the specificity.We performed in vitro transcription to obtain GETV RNA,using the recombinant plasmid containing the primer probe design region as a template.The plaque formation experiment was carried out to determine the virus titer of GETV(GZ0885 strain).Select Sindbis virus,Japanese encephalitis virus and other closely related or common arboviruses for simultaneous detection to evaluate the specificity of the method.We serially diluted the titrated virus and extracted nucleic acid as a template,or serially diluted in vitro transcribed RNA as a template.Then,for evaluating the sensitivity and stability of the method,we perfomed a TaqMan real-time fluorescent quantitative RT-PCR to construct a standard curve.We used the established detection method to conduct GETV screening in batches on 12,634 mosquitoes collected in Hebei Province,Hunan Province,Fujian Province,and Chongqing city from 2009 to 2014 and 12,731mosquitoes collected in Ningxia Hui Autonomous Region in 2019 were screened in batches for GETV to understand the recent distribution of GETV in China,which provides a basic basis for disease prevention and control.Results:NCBI Blast showed that the primers and probes designed for the relatively conservative ns P4 gene has no match with other arboviruses,which indicates that the primers and probes have good specificity.Through in vitro transcription,we obtained the GETV RNA with a concentration of 1.85×10⁹ copies/?l.Through plaque formation experiments,we found that the titer of the GZ0885 strain kept in the laboratory was 10^6.36PFU/ml.This method can specifically detect GETV and has no cross-reactivity with a variety of arboviruses.The goodness of fit R² of the standard curve with GETV extracted nucleic acid and in vitro transcription RNA as templates is between 0.99 and 1,indicating a good linear relationship;the amplification efficiency Eff is between 90%and 110%,which indicates that this method has relatively complete amplification and can accurately reflect the RNA concentration.This method has good sensitivity,and the detection limit can reach1.0×10 pfu/ml or 1.0×10² copies/reaction.The intra-assay and inter-assay coefficients of variation are both less than 5%,indicating that the method has good stability.In addition,There was 1 case GETV positive in 196 batches of mosquitoes collected from Hunan Province,Hebei Province,Fujian Province and Chongqing city and 7case GETV positive in 189 batches of mosquitoes collected from Ningxia Hui Autonomous Region.The GETV positive rate reached 3.70%,which shows that this method is feasible.Conclusion:In summary,this study successfully established a TaqMan probe real-time fluorescence quantitative RT-PCR method,which can be used for GETV screening.This method has the advantages of high sensitivity,strong specificity,and good stability.In this study,GETV was detected in mosquito samples from Ningxia Hui Autonomous Region in 2019,indicating that GETV has been distributed in Ningxia Hui Autonomous Region.In addition,GETV was also detected in mosquito samples from Hebei Province in previous years,suggesting that a GETV cycle has already existed in Hebei Province.