Establishment Of QPCR Detection Method For Asian Zika Virus And Bioinformatics Research Of B Cell Epitope Based On NS1 Gene

Zika virus(ZIKV)is an RNA virus transmitted by mosquito bites.It was first discovered in 1947 in the fever rhesus monkeys in Uganda.According to the genotypes differences,it can be classified into two types:African type and Asian type.Although ZIKV was discovered early,it has not attracted worldwide attention because of its mild clinical symptoms,such as rash,fever,joint pain,conjunctivitis and so on.Until 2015,the outbreak of the Brazilian region,and gradually presented many serious complications,including neonatal microcephaly,Guillain-Barre syndrome,etc.Then the World Health Organization listed Zika as a global public health emergency.Therefore,it is particularly important to establish and perfect the early detection and pathogen classification system of Zika virus.In this study,we first analyzed the characteristics of the Asian Zika virus genome sequence,and used AlleleID7.2 software to design specific probes and primers to establish a fluorescent quantitative PCR detection method for Asian Zika virus,and the standard curve was obtained.The feasibility of the method was verified by specificity test,sensitivity test and repeatability test.Subsequently,using the web server and a variety of bioinformatics analysis software,systematic bioinfonnatics analysis of Zika virus NS1 protein,including protein antigenic properties,transmembrane domain and signal peptide prediction,secondary structure and hydrophobicity.Many parameters such as antigenicity,flexibility and surface accessibility were predicted,and the epitopes of B cell dominant antigen were screened from a large number of protein antigen data,which provided a theoretical basis for the development and development of vaccines in the future.Detection of designed PCR primers and probes were of good specificity,with the lower limit of detection being 3.415 copies/?L,and there were no cross reactions between the African Zika virus,type 1-4 dengue virus,Hepatitis C virus,Chikungunya virus and Japanese encephalitis virus.The standard curve was successfully constructed,and the relevant coefficient was 1,the amplification efficiency is as high as 102%;The repeatability experiment results show that the coefficient of variation of CT value in each group is less than 2%,which indicated that the stability and repeatability were good.At the same time,through the multi-software,multi-parameter verification,the two optimal B cell epitope peptides were determined from the five preliminary epitope peptides,which were located at positions 253-266(HNTREGYRTQMKGP)and 333-345(MEIRPRKEPESNL).The fluorescent quantitative PCR technology of Asian Zika virus established in this project is characterized by high efficiency,specificity and high sensitivity.It can quickly identify Asian Zika virus strains,which can be used inthe etiologic typing,early diagnosis and prevention of Asian Zika Virus.NS1 protein is the main protein antigen,which can stimulate the body to produce specific antibodies,which can be used as an early serum marker for viral infection.Therefore,the prediction of antigen epitopes is of great significance to the pathogenesis of infected patients and the development of vaccine.