The Establishment Of DdPCR And RT-qPCR Methods And The Analysis Of B Cell Linear Epitope For ZIKV

BackgroundsZika virus(ZIKV)is a singkle-stranded RNA virus belonging to the family Flaviviride genus Flavivirus.It was first isolated in Uganda in 1947 in monkeys through a network that monitored yellow fever(Dick et al.,1952)and transmitted through the bite of an infected mosquito from the Aedes genus,mainly Aedes aegypti in tropical regions(Weaver et al.,2016).In humans,ZIKV disease is characterized by mild fever(37.8-38.5℉),joint pain(particularly pain in the hands and feet),myalgia,headache,orbital pain,conjunctivitis,and skin rash and can also cause microcephaly and Guillain-Barre syndrome(Chouin et al.,2016).ZIKV encodes three structural proteins(capsid protein C,precursor protein prM,and envelope protein E)and seven nonstructural proteins(NS1,NS2A,NS2B,NS3,NS4A,NS4B,and NS5)(Fontes et al.,2017).There are two main method for the detection of ZIKV,such as host serological tests and nucleic acid based methods.Due to the cross-reacting antigens between ZIKV and other flaviviruses,the former one may generate false positive results in the co-existing prevalent.Real-time quantification PCR(RT-qPCR),the standard tool to quantify virus DNA or RNA currently,is limited when detecting serum samples with low virus concentration(Balm et al.,2012).Micro-droplet digital polymerase chain reaction(ddPCR)is a new molecular method enabling absolute quantification of DNA targets without a calibration curve as used commonly in RT-qPCR.The recent development of ddPCR has been used widely in medical researches and clinical applications,but still not been applied to the detection of ZIKV yet.The first part of this article established ddPCR and RT-qPCR protocols for the detection of ZIKV based on the NS5 gene and evaluated the sensitivity,specificity and repetitiveness of them,aiming to provide a more accurate method for the detection of ZIKV samples with low virus concentration or early diagnosis.Vaccination is the most effective way to prevent ZIKV infection in the absence of antiviral drugs.However,there is no approved Zika vaccine currently,and the development of Zika vaccine is imminent.The B cell epitope is a chemical group that specifically recognized and bound on the surface of an antigen molecule by a B cell receptor(BCR)or an antibody Fab,and is a material basis for humoral immunity.As the core part of the antigen,the B cell epitope is the focus of research on immune diagnosis and immunotherapy.It can save a lot of laboratory workload and avoid the blindness of traditional epitope research.Various studies have shown that epitope-driven vaccines can effectively stimulate protective immune responses against a variety of pathogens,such as influenza virus,human immunodeficiency virus,hepatitis B virus and hepatitis C virus.The identification of B cell epitopes is important for the development of epitope vaccines.Immunoinformatics is becoming more and more common in the field of vaccine development.The use of genomic and proteomic information in this field provides much information to predict potential vaccine candidate.The second part of this article used the protein sequence of ZIKV strain Z16006(accession:AMR39830.1)in the B cell linear antigen epitopes prediction,including antigenicity,basic physicochemical properties and secondary structure analysis.After screening,we found some of dominant epitopes in the B cell of ZIKV finally.The multi-parameter multiple methods were used to predict the B cell epitope of Zika virus,which laid the theoretical foundation for the further experimental identification of its epitope and preparation of monoclonal antibody.ObjectivesIn this study,we established micro-droplet digital polymerase chain reaction(ddPCR)and real-time quantification PCR(RT-qPCR)protocols for the detection of ZIKV based on the NS5 gene.And we evaluated the sensitivity,specificity and repetitiveness of them.Also We used bioinformatics methods to obtain the potential B cell antigen epitopes of ZIKV protein.MethodsWe designed and evaluated the primers and probes based on NS5.And the preparation of plasmid for standards curves were also performed so that the methods of RT-qPCR and ddPCR were established.Then we established and evaluated the sensitivity and specificity of them.For B cell linear antigen epitopes prediction,the protein sequence of ZIKV strain Z16006(accession:AMR39830.1)was used.First obtained the protein sequence of Z16006 from NCBI,using the online software VaxiJen v2.0 to evaluate the antigenicity score.Then the basic physical and chemical properties were also analyzed in ProParam.Utilizing SOPMA to performed the secondary structure.Finally the analysis for the B cell antigen epitopes was subsequently predicted by IEDB and ABCpred.Results1.For the ZIKV standard plasmid,RT-qPCR method showed that the cycle threshold value(Ct)was linear from 101 copy/μL to 108 copy/μL molecules,with R2=0.999 of the standard curve and the amplification efficiency 92.203%,but the concentration of 1 copy/μL molecule could not be detected.In comparison with RT-qPCR,ddPCR method had the linear range of 101-104 copy/μL molecules,and could detect low concentration samples with 1 copy/μL molecule.For detecting ZIKV from clinical samples,RT-qPCR was a better choice for higher concentration samples(above 101 copy/μL),while ddPCR has excellent accuracy and sensitivity for low copy samples.The results indicated that ddPCR method had a great significance in early diagnosis,lab research,and health monitoring.2.We acquired 52 potential B cell antigen epitopes.Conclusions1.A good linear relationship could be obtained in RT-qPCR when detecting samples with higher concentrations(above 101 copy/μL),but this method is not suitable for detecting low copy(less than 101 copy/μL).DdPCR detection at low concentration samples,especially serum sample,shows excellent specificity and sensitivity;however,it cannot be applied to high concentration samples.Therefore,RT-qPCR and ddPCR could complement each other in clinical examination.In clinical samples with low concentration ZIKV,for example,in samples obtained 1-3 days after infection,ddPCR can show the best diagnostic accuracy and sensitivity.For routine laboratory virus nucleic acid detection,including the analysis of clinical samples obtained after 3 days of infection,RT-qPCR can be used.2.The bioinformatics software SOPMA was used to predict the secondary structure of the 216006 protein,and the B-cell phenotypic epitopes were obtained through comprehensive prediction analysis by the IEDB and ABCpred software.Through this study,the bioinformatic properties of Z16006 protein were clarified,which provided the basis for the identification and screening of dominant epitopes.It meanwhile laid the theoretical foundation on immunoprevention and treatment development of epitope vaccine for ZIKV.