Study On The Detection Of Hantavirus By Hypersensitive Single-molecule Fluorescence Localization

Objective: Hemorrhagic fever with renal syndrome(HFRS)is a viral disease caused by Hantavirus,which is widespread in the world and seriously threatens human life and health.In order to achieve early detection and treatment,it is very important to strengthen the detection of Hantavirus.At present,a variety of detection methods have been reported,but they still have many shortcomings,including complex detection procedures,time consuming,large sample size,not suitable for low abundance analysis and other defects.Fortunately,with the development of fluorescence microscopy and spectroscopy,single-molecule fluorescence detection has been successfully applied to the detection of various biomarkers.Compared with traditional technology,single-molecule fluorescence detection has the advantages of less sample consumption,high sensitivity and fast response time,so it is an ideal detection platform for constructing ultra-sensitive biosensors.However,the signal amplification strategy still has some problems,such as weak fluorescence signal,instability and complex operation.Therefore,this work aims to develop a simple and stable single-molecule fluorescence detection method with high fluorescence intensity to achieve the direct detection of Hantavirus RNA.Methods: In this paper,a single-molecule detection method based on multi-fluorescent nucleic acid probe was constructed.5-acetyne-2 ’deoxyuracil nucleoside was used to replace deoxythymidine nucleoside for polymerase chain reaction to synthesize multi-alkyne DNA chain.According to the acetylene azide click chemical reaction catalyzed by copper,the azide Cy5 fluorescent molecule was connected to the multi-alkyne DNA chain to synthesize the multi-alkyne DNA chain.The reporter unit was assembled into a magnetic bead-capture probe-polyfluorescent nucleic acid probe to identify Hantavirus-specific nucleic acid sequences,which were then quantitatively detected by single-molecule fluorescence localization imaging.Results: The fluorescence intensity of the synthesized multi-fluorescent DNA nucleic acid probe was significantly improved,which was about 9 times that of the commercial single fluorescent DNA strand.The detection platform developed on the basis of the probe was optimized through a series of optimization,such as the concentration of the captured probe,the volume of replacement reaction,buffer solution and time,etc.,and the platform realized the high selectivity and high sensitivity detection of Hantavirus nucleic acid sequence,and the detection limit was up to 41.86 a M.The detection system can still detect Hantavirus RNA with high sensitivity in simulated serum complex samples.Conclusions: The single-molecule fluorescence localization imaging method based on multi-fluorescent DNA strand can detect Hantavirus RNA with high sensitivity and high specificity,and this method has great potential in early clinical diagnosis.