Research On Detection Of Dengue Virus Based On Hairpin Selfassembly Exponential Amplification Biosensor

Objective:Dengue is a endemic infectious disease in tropical and subtropical regions,which was transmitted by mosquito bites.Different dengue virus（DENV）strains can be divided into four dengue virus serotypes（DENV 1-4）according to plaque reduction neutralization test and each serotype is contagious.Dengue is characterized by fever,rash and bleeding.Clinical examination methods for dengue include blood routine,urine routine,virus isolation,serum immunology and so on.However,these detection methods are usually time-consuming and high-cost.Therefore,a rapid,simple and inexpensive detection strategy of DENV is crucial for early diagnosis of dengue.In this study,we propose an isothermal,enzyme-free exponential signal amplification strategy based on CHA combines with the spatially sensitive fluorescence signal of pyrene excimers,achieving a rapid and early detection for dengue virus.Methods:This strategy involves four types of oligonucleotides:three pyrene modified hairpins（H1,H2 and H3）and one catalyst DNA（C）.In the absence of C,the hairpin structure maintain that spatially separate the pyrene molecules at the 3′and 5′ends and avoid the formation of an excimer.However,Upon the introduction of C,CHA probes undergo a series of TMSD reactions to generate intermediates（I）and the formed pyrene excimers were emitted at 485 nm.Moreover,we introducedγ-CD with lipophilic inner cavity and hydrophilic outer surface to further enhance the fluorescence signal intensity of excimer.Notably,the catalytic region（a*-x*-b*-y*）of catalyst DNA（C）used to activate the CHA with the help of hairpin H4,is completely independent of the target sequence.Therefore,this strategy is applicable to detect a variety of targets,such as nucleic acids,aptamers,small molecules and so on.Rapid detection of dengue virus can be achieved by monitoring changes in fluorescence intensity.Results:In this work,we design an exponential amplification strategy based on catalytic hairpin assembly（CHA）with the spatially sensitive fluorescence signal of pyrene excimers.In the absence of dengue-specific conserved RNA,the fluorescence signal of pyrene activators is weak.When dengue conserved RNA was added,the fluorescence signal was strongest within 50 minutes.The fluorescence intensity was log-linear correlation with DENV-1 RNA concentration in 0.1-50 n M（R²=0.99）with the linear equation of F=935+508 lg C_{（n M）}.The corresponding detection limit is0.048 n M according to the measured results.Low concentrations of dengue can be detected.To further investigate the specific detection of DENV1 RNA,three other dengue virus serotypes（DENV2-4）were selected as interfering nucleic acids.The fluorescence intensity produced by（DENV2-4）RNA was almost the same as that produced by the background of system,while the fluorescence intensity generated by DENV-1 RNA was significantly higher than them,which fully demonstrated the high selectivity of the new system for DENV-1 RNA detection.Conclusions:In this paper,an enzyme-free,isothermal,rapid exponential signal amplification biosensor platform was established through a series of optimization experiments,feasibility experiments and specimen recovery experiments.The platform enables early,rapid detection of dengue viruses.It plays an active role in the development and development of novel RNA diagnostic methods for dengue virus.