Establishment And Application Of The Rt-hemi-nested PCR Assay For Mosquito-borne Virus

BackgroundMosquito-borne virus can be transmitted through mosquito biting on sensitive vertebrates and can cause natural focus infection disease worldwidely.There are as many as 260 viruses isolated from mosquitoes,Japanese encephalitis virus(JEV),Dengue virus(DENV),West Nile virus(WNV),Yellow fever virus(YFV)of flavivirus,Chikungunya virus(CHIKV)and Sindbis virus(SINV)of alphavirus are predominant mosquito-borne viruses.All of them can cause human diseases,such as fever,hemorrhage,rash,joint pain and encephalitis,and serious cases can lead to death.Mosquito-borne diseases have beco-me a serious public health problem worldwidely.Except for Japanese encephalitis virus and yellow fever virus,there are no effective vaccines and treatments for other mosquito-borne viruses.Therefore,it is of great significance to establish a simple,rapid,specific and sensitive detection method for the prevention and control of mosquito-borne diseases.ObjectiveRT-hemi-nested PCR based on RT-PCR technology was established in this study,which can be used to detect mosquito-borne flavivirus and alphavirus respectively,and provide technical support for early and rapid diagnosis of mosquito-borne viruses.Methods1.Primer designThe complete genome sequences of mosquito-borne flavivirus and alphavirus were downloaded from Gen Bank database.Conservative sequences were obtained by comparing and analyzing the viruses with bioinformatics software.Primers were evaluated by NCBI blast and the most suitable primers were selected.2.Preparation of the standard samplesThe PCR product was purified and then recombined into plasmid.The plasmid was 10times diluted repeatedly to make the virus standard samples.The virus standard sample was used as the template for RT-hemi-nested PCR detection.3.Optimization of the reaction systemsThe two reaction systems of mosquito-borne flavivirus and alphavirus were optimized to reduce the occurrence of non-specific bands by changing the concentration of primers and annealing temperature.4.Evaluation of specificity and sensitivity of RT-hemi-nested PCR assayThe designed flavivirus and alphavirus primers were used to detect mosquito-borne flavivirus and alphavirus respectively to verify specificity.The established RT-hemi-nested PCR methods were used to detect viral standard samples with different dilutions,and the sensitivity of the detection system was evaluated.5.Detection of mosquito specimens collected in the fieldTwo RT-hemi-nested PCR detection systems were established to detect mosquito specimens collected in the field.RNA was extracted from mosquito specimens by RNA extraction kit,and c DNA was obtained by reverse transcription kit.Mosquito-borne flavivirus RT-hemi-nested PCR and mosquito-borne arbovirus RT-hemi-nested PCR were used to detect the virus,respectively.The positive samples were sequenced to determine the type of virus.Results1.The specificity detection results in this study showed that only the target bands appeared in the flavivirus and no bands appeared in control,indicating the high specificity in detecting mosquito-borne flavivirus.The sensitivity test results showed that the lowest detection limit of JEV,DENVII,YFV and WNV was 3×10~4copies/?l,3×10~6copies/?l,3×10~5copies/?l and 3×10~4copies/?l,respectively.However,the lowest detection limit of regular-PCR for JEV,YFV and WNV was 3×10~7copies/?l,3×10~9copies/?l and 3×10~7copies/?l.Dengue virus has never been detected by regular-PCR.Therefore,the sensitivity of hemi-nested PCR is 10~3-10~4 times higher than regular-PCR.In conclusion,this method greatly improves the sensitivity and reduces the possibility of missed detection of regular-PCR.2.The specificity detection results showed that only the target bands appeared in the alphavirus and no bands appeared in control,indicating the high specificity of the mosquito-borne alphavirus RT-hemi-nested PCR.The sensitivity test results showed that the lowest detection limit of SINV and CHIKV was 3×10~3copies/?l and 3×10~4copies/?l,while the lowest detection limit of regular-PCR for SINV and CHIKV was 3×10~7copies/?l and 3×10~8copies/?l,respectively.Therefore,the sensitivity of hemi-nested PCR is 10~4times than that of regular-PCR.3.Mosquito-borne flavivirus and alphavirus RT-hemi-nested PCR methods were used to detect mosquito specimens collected in the field.Ninety-six groups of mosquito specimens were used for detection of flavivirus and alphavirus.Two positive results were detected and they are genotype I Japanese encephalitis virus and genotype III Japanese encephalitis virus based on the results of sequencing and identification.Mosquito-borne alphavirus was not detected in all of the 96 groups of samples,which may be contributed to the species of mosquito used in this study.Becaused Aedes are the main vectors of alphavirus such as Chikungunya virus and Sindbis virus,while most samples in this study were Culex and Armigeres obturbans.Conclusions1.RT-hemi-nested PCR for mosquito-borne flavivirus was successfully established in this study.This method has better specificity,higher sensitivity and can detect multiple viruses at the same time.2.RT-hemi-nested PCR for mosquito-borne alphavirus was successfully established in this study.The method is characterized by better specificity,higher sensitivity,more simple and rapid.Meantime,this method is easy to get popularization,which would be helplful in large-scale epidemiological investigation.