Development Of A Two-tube Multiplex Quantitative Real-time Polymerase Chain Reaction To Detect Six Arboviruses

Background and Objective:Chikungunya virus?CHIKV?,dengue virus?DENV?,West Nile virus?WNV?,yellow fever virus?YFV?,Japanese encephalitis virus?JEV?and Zika virus?ZIKV?are common six arboviruses,which are mainly spread by mosquito bites.They are widely distributed in the world and have large numbers of infections,causing a serious disease burden.Humans infected by these viruses displayed similar clinical manifestations such as fever,rash,and fatigue and other similar clinical manifestations,so it is difficult for clinicians to identify the disease in the early stage.In addition,the emergence of co-infected cases poses great challenges for clinical diagnosis and treatment,and can easily lead to missed diagnosis and misdiagnosis.At present,the widely used detection methods cannot fully meet the clinical needs due to various factors.Therefore,the development of a new differential diagnosis method is of great significance for the prevention,diagnosis,treatment and control of such diseases.The quantitative real-time PCR developed in recent years has the characteristics of strong specificity,high sensitivity and good repeatability,and has been widely concerned.In this study,quantitative real-time PCR was used to establish a method for simultaneous detection of the above six arboviruses in two tubes,which greatly improved the detection efficiency and provided a new rapid detection method for clinical diagnosis.Methods:The sequences of CHIKV,DENV,WNV,JEV,ZIKV and YFV were downloaded from the National Center for Biotechnology Information?NCBI?database,and sequence alignment analysis was performed using MEGA7.0 software.Primers and probes were designed for the conserved regions of the CHIKV-E3,DENV3'-UTR,WNV-NS2A,YFV5'-UTR,JEV5'-UTR,ZIKV-E genes.The standard curve was established by the standard plasmid and viral RNA templates and was then used to evaluate the specificity,sensitivity,and reproducibility for this method.Results:The designed primer probes did not cross-non-specific amplification with each other.The detection limit of detection of CHIKV,DENV,WNV,YFV,JEV,ZIKV was 5 copies/reaction and 10^-3?10^-1TCID₅₀/reaction;the correlation coefficient between the cycle threshold?Ct?value and logarithm of the of the template copy numbers or the virus titers is above 0.99.Repeatability tests were performed using standard dilution plasmids of different dilution gradients,and the coefficient of variation of Ct values in each dilution was less than 4%.Then 31 clinical samples from suspected cases were detected by the developed method with a positive rate of 87.1%?27/31?,which was consistent with the test results of the commercial kit,and the coincidence rate was 100%.There were no co-infections in these samples.Using this method to detect samples can be completed in as little as 2 hours.Conclusions:We have successfully established a quantitative real-time PCR method that can detect CHIKV,DENV,WNV,YFV,JEV and ZIKV at the same time,and has the advantages of good specificity,high sensitivity and stability,fastness and simplicity.The method can be used for clinical sample detection and has certain practical value.