Development And Evaluation Of Microfluidic Real-time Quantitative RT-PCR Detection Methods For Common Hemorrhagic Fever Viruses

Objective To develop rapid nucleic acid detection methods for common hemorrhagic fever viruses,based on microfluidic and fluorescence quantitative RT-PCR technologies,including Severe fever with thrombocytopenia syndrome virus(SFTSV),Rift Valley fever virus(RVFV),Heartland virus(HLV),Zika virus(ZIKV),Dengue virus(DENV),Yellow fever virus(YFV),Chikungunya virus(CHIKV),Seoul virus(SEOV),Hantaan virus(HTNV),Sudan Ebola virus(SEBOV),Zaire Ebola virus(ZEBOV),Marburg virus(MARV)and Lassa virus(LASV).In order to achieve faster detection speed than traditional fluorescent quantitative RT-PCR detection methods and provide reference for the rapid diagnosis of these 13 virus infections.Methods Specific primers and probes were designed targeting the NP gene of SFTSV,RVFV,HLV,HTNV,SEOV,ZEBOV,SEBOV and MARV,the NS1 gene of ZIKV,the NS5 gene of DENV and YFV,the E1 gene of CHIKV,respectively.In vitro transcribed RNA standards of these 13 virus were prepared and used to evaluate the sensitivity of the methods.The specificity of the methods were evaluated by using Japanese encephalitis and other related viruses.Simulated positive samples were prepared by adding RVFV,HLV,ZIKV,YFV,CHIKV,HTNV,SEOV,ZEBOV,SEBOV,MARV,LASV pseudoviruses or virus cultures into healthy human serum,which were used to verify the detection methods and compared with traditional fluorescent quantitative RT-PCR detection methods.DENV detection methods were validated using clinicalpatient specimens and compared with traditional fluorescent quantitative RT-PCR detection methods.Results Microfluidic real-time fluorescence quantitative RT-PCR detection methods of SFTSV,RVFV,HLV,ZIKV,DENV,YFV,CHIKV,HTNV,SEOV,ZEBOV,SEBOV,MARV and LASV were successfully established.In vitro transcribed RNA standards for 13 types of viruses were successfully prepared.The limit of detection(LOD)of these detection methods were 61.4 copies/ PCR,60.9 copies/ PCR,156.9 copies/ PCR,and 30.9 copies/ PCR,226.2 copie/ PCR,19.8 copies/ PCR,535.6 copies/ PCR,119.1 copies/ PCR,123.6 copies/ PCR,67.8 copies/ PCR,12.8 copies/ PCR,61.2 copies/ PCR,11.3 copies/ PCR,except that the detection limit of CHIKV was slightly high,the other detection methods showed satisfactory sensitivity.Thirteen virus detection methods have no cross-reactivity with other viral nucleic acids.Simulated clinical samples of RVFV,HLV,ZIKV,YFV,CHIKV,HTNV,SEOV,ZEBOV,SEBOV,MARV,and LASV were prepared by using virus cultures or pseudoviruses mixed with healthy human serum.Twelve virus microfluidic real-time quantitative RT-PCR detection methods except SFTSV were used to verify corresponding simulated samples or clinical patient specimens.The coefficient of variation of the Ct value of the simulated sample verification results was about 2 %.The detection rates of the simulated sample verification and clinical patient specimen verificationwere consistent with traditional real-time fluorescent quantitative RT-PCR detection methods,all of which were 100 %.Thirteen virus microfluidic real-time fluorescent quantitative RT-PCR detection methods were used to verify the collected healthy human serum samples,and the results were consistent with the real-time fluorescent quantitative RT-PCR detection methods,all of which were negative.Discussion The SFTSV,RVFV,HLV,ZIKV,DENV,YFV,CHIKV,HTNV,SEOV,ZEBOV,SEBOV,MARV and LASV microfluidic quantitative RT-PCR detection methods showed satisfactory specificity and stability.Except that the detection limit of CHIKV was slightly high,the other detection methods showed satisfactory sensitivity.Except for SFTSV,which had not been verified,the detection effects of other virus detection methods on simulated samples or clinical samples were consistent with the traditional real-time fluorescence quantitative RT-PCR detection method.The detection could be completed within 25 minutes,and the detection speed is faster than the traditional real-time fluorescent RT-PCR detection method,which could be used for laboratory detection and early diagnosis.