Experimental Study On Rapid And Combined Detection Of Four Common Mosquito-borne Viruses By Multiplex Recombinase Polymerase Amplification

Objective On the basis of Recombinase Polymerase Amplification（RPA）,to establish the single and quadruple reverse transcription recombinase polymerase amplification methods（RT-RPA）for the diagnosis and differential diagnosis of dengue virus,yellow fever virus,chikungunya virus,Zika virus infection in a single reaction.Methods ①Design and screen primers and probes corresponding to dengue virus（DENV）,yellow fever virus（YFV）,chikungunya virus（CHIKV）and Zika virus（ZIKV）,then the RT-RPA system was established to detect the plasmid standards of DENV,YFV,CHIKV and ZIKV,and the specificity,sensitivity and repeatability were evaluated.② On the basis of RT-RPA reaction,orthogonal design was used to optimize different reaction components,then the quadruple RT-RPA system was established to simultaneously detect the plasmid standards of DENV,YFV,CHIKV and ZIKV,and the specificity,sensitivity and reproducibility were evaluated.③ The established RT-RPA and quadruple RT-RPA reaction systems and their specificity,sensitivity and reproducibility were verified by in vitro transcribed RNA and clinical serum samples.At the same time,the differences in the detection rate of positive samples by RT-PCR,single RT-RPA and quadruple RT-RPA were compared.Results①The RT-RPA and quadruple RT-RPA established in this experiment can be used for the detection of plasmid standards,in vitro transcribed RNA and clinical samples of dengue virus,chikungunya virus,yellow fever virus and Zika virus.② The RT-RPA and quadruple RT-RPA established in this experiment have no cross-reaction with common pathogens.The detection limit of RT-RPA for DENV,ZIKV,CHIKV,YFV standard plasmid DNA are 1×102 copies/ml,1×102 copies/ml,1×102 copies/ml,<1×102 copies/ml.The detection limit of DENY,CHIKV,YFV,ZIKV in vitro transcribed RNA diluted with water for injection are 5×102 copies/ml,5×102 copies/ml,5×102 copies/ml,5×103copies/ml,and of DENV,CHIKV,YFV,ZIKV in vitro transcribed RNA diluted with normal human serum are 5×103 copies/ml,5×103 copies/ml,5×103 copies/ml,5×103copies/ml.③There was no significant difference in the detection rate of positive samples among RT-RPA,quadruple RT-RPA and RT-PCR（P=0.972）.Conclusion The RT-RPA and quadruple RT-RPA assays established in this study can be used for the detection of plasmid standards,in vitro transcribed RNA and clinical samples of DENV,CHIKV,YFV and ZIKV.The detection methods have good specificity,high sensitivity and good repeatability,and the detection rate of plasmid standards,in vitro transcribed RNA and clinical samples has no significant difference with RT-PCR.It plays an important role in the early diagnosis and differential diagnosis of mosquito-borne infectious diseases.It can also provide research directions for the establishment of rapid diagnosis methods for other emerging and recurrent infectious diseases.