Construction Of A-PCR Combined With AuNPs@SiO₂ For Paper-based Detection Of Ebv Nucleic Acids

Objective:EBV is a linear double-stranded DNA virus of the B-lymphophilic subgroup of the gamma family of herpesviruses that latently infects B-lymphocytes and was the first oncogenic virus discovered in humans.Current research has found that EBV is associated with the development of a variety of diseases.Some of these methods are sensitive and specific,but cumbersome and expensive to perform,while others are rapid and simple,but have a low positive detection rate.Therefore,the construction of a simple,rapid and sensitive test is essential for the detection of EBV infection.A-PCR is simple to perform and can obtain a large amount of single-stranded target DNA.AuNPs@SiO₂composite particles are stable and can load a large amount of capture probes.The paper-based assay is simple and low cost.The combination of these three materials and techniques allows for a simple,low cost and stable EBV detection system.Methods:In this study,a novel paper-based lateral flow nucleic acid（LFNA）detection platform was developed using asymmetric polymerase chain ceaction（A-PCR）for signal amplification.This new method allows the visual detection of EBV nucleic acids with high specificity and low cost.In addition,a capture probe（CP）/gold nanoparticles（AuNPs）and silicon dioxide（SiO₂）,（AuNPs@SiO₂）nanosphere/target DNA/avidin complex sandwich system is used as a sensing platform as part of the assay.Biotin-labelled target DNA was obtained by A-PCR and then introduced into the LF device.The CP/target DNA/AuNPs@SiO₂complex is captured into the test region by specific reaction of biotin with affin,and the remaining CP/AuNPs@SiO₂particles are captured into the quality control region by hybridisation of CP with a quality control probe.AuNPs@SiO₂is enriched in the test and quality control regions of the NC membrane to obtain visual detection of specific target sequences.Results:A large number of single-stranded target sequences containing biotin were amplified by A-PCR using a large number of simplex primers,and a final upstream and downstream primer ratio of 1:50 was selected by characterisation and optimisation by agarose gel electrophoresis.The AuNPs@SiO₂composite particles were successfully prepared by combining amino groups on the SiO₂surface with hydrogen ions on the colloidal gold surface,and were characterised and optimised using UV spectrophotometry and transmission electron microscopy.Actual samples were assayed on a paper-based platform and the amount of capture probe and affinity concentration were optimised.A capture probe dosage of 60μl and an affinity dosage of 1ng/ml were chosen and the reproducibility of this system was tested with a coefficient of variation of 0.087,which is reproducible.The detection limit of this method is 50 n M,which is lower than that of the LFNA biosensor without PCR amplification and without AuNPs@SiO2 particles（LFNAB）.Conclusion:In summary,the paper-based novel detection platform constructed in this study is a low-cost,efficient and rapid visual detection method,and also provides ideas for the construction of new methods for nucleic acid detection of other pathogens.