Development And Preliminary Evaluation Of The Nucleic Acid Detection Method Of Viruses Related To Zika Virus

Zika virus is classified in flaviviridae family,they can easily cause nerve arbovirus of systemic disease.It has been transmitted by mosquito-borne.Like ZIKV,Dengue virus of arboviruses and Chikungunya virus are classified in togaviridae,they all spread through mosquito.They all have the same clinical symptoms and endemic areas.So it's crucial to establish the nucleic acid detection method for triplex real-time RT-PCR assays for three viruses in filed tests.The real-time RT-PCR method has high sensitivity,specificity and consistency,it can achieve detection of multiple unknown pathogens.We have designed the primers and probes of ZIKV,DENV,CHIKV and optimized the reaction system,then estabilished the triplex real-time RT-PCR assays.We have evaluated the sensitivity,specificity and consistency of this method with in vitro transcribed RNA of three viruses.Then estabilished the standard curve with gradient dilution templates and calculated the LOD of DENV.The LOD of four types of DENV respectively are 9Copies/PCR,9Copies/PCR,41Copies/PCR,4Copies/PCR and the LOD of CHIKV and ZIKV are respectively 3Copies/PCR and 15Copies/PCR.We also establish the standard curves with diluted series of viruses.The LOD of four types DENV,CHIKV and ZIKV are respectively 2PFU/ml,9PFU/ml,1PFU/ml,4PFU/ml,2PFU/ml and 6PFU/ml.Compared with monoplex method,there is not much difference between monoplex and triplex methods,it also has high sensitivity.To assess the specificity of this method,we use 11 kinds of viral nucleic acid to detect,it turns out there is no cross reaction;There were 20 repetitions of three kind of high,medium and low concentrations of the three viruses,the standard deviation was controlled within 0.5,and the variation coefficient was below 2%,it proves a good stability.There are 20 serum samples of patients with DENV acute infection and the clinical simulation samples of ZIKV and CHIKV,it has good positive rate.In addition,our research assembles the templates of three kind of virus and certify,the results showed that single template,double templates and three templates samples can accurately been detected and there was no significant difference of CT value with the same virus.Our study aims to establish an RPA isothermal amplification method for rapid field detection in ZIKV outbreak.By analyzing ZIKV sequence alignment,we have choosed nine upstream primers and fifteenth downstream primers,we use Basic-RT reaction system amplification,and identification by agarose gel electrophoresis.We have selected 2 upstream primers and 4 downstream primers,then we got eight single bright stripe by garose gel electrophoresis.Based on ZIKV NS1 fragment sequence,we have designed multiple primers and probes after rounds of selection,and optimize the reaction system.the results showed that the 2-1 assays of primers and probe(ZIKV-EXO-F1-4?ZIKV-EXO-R2-3-1?ZIKV-EXO-P1-4)has a good expansion.And based on the combination of primers and probes,we optimized other factors influencing the amplification reaction system,such as magnesium concentration,the concentration of primer and probe,the choose of reverse transcriptase,RNase Inhibitor conditions.Finally we estabilish the method of real-time RPA technology and evaluate its sensitivity,specificity and stability.lt proves that LOD of ZIKV is 200 Copies/PCR,and there is no cross reactions with other viruses,it has a good specificity and repeatability.The clinical serum specimens of ZIKV patients can be detected 100%,and the Ct value of the samples is about 16.07 to 27.05.Therefore,our research has estabilished triple real-time RT-PCR technology to simultaneously detect the pathogens and real-time RT-RPA technology based on ZIKV,this experiments have huge potentials in the future.