Study On The Detection Of Norovirus Nucleic Acid Based On Enzyme-free Isothermal Signal Amplification Technology

As one of the main pathogens causing non-bacterial food poisoning,Norovirus usually inhabits shellfish,berries and other foods.It is called"intestinal flu"on account of high infectiousness.Recently,the epidemic caused by food-borne Norovirus has been growing rapidly,so it’s essential to develop rapid and effective detection methods.Nucleic acid-targeted molecular biology technique such as PCR is a very important type of detection method in virus detection.Isothermal signal amplification technology has high sensitivity comparable to PCR method and free of variable temperature cycling process as well as temperature control equipment.Enzyme-free isothermal signal amplification technology that has been developed subsequently has the advantages of easy operation and high efficiency,which has attracted researchers’attention in recent years and been applied to the construction of biosensors in the fields of food safety,environmental monitoring,and medical diagnosis.In this paper,two new types of enzyme-free isothermal signal amplification methods were constructed by using GⅡNorovirus conserved sequence as the detection target nucleic acid sequence.Firstly,a detection method of nucleic acid based on seesaw-gate driven isothermal signal amplification technology（SGA）was constructed;Secondly,silver clusters was used as label-free signal output,catalytic hairpin self-assembly amplification（CHA）technology in combination of magnetic ball separation,a nucleic acid detection method based on magnetic separation and CHA was constructed.The main research contents of this paper are followed:1.Established an isothermal signal amplification technology based on circular seesaw gate.This technology was based on toehold-mediated strand displacement reaction and realized circular amplification of the fluorescent signal through a series of reactions similar to seesaw reaction.On this basis,a highly sensitive enzyme-free nucleic acid detection method has been established,which had the advantages of simple sequence design,gentle reaction system and convenient operation.After optimization of the sequence concentration,reaction temperature and reaction time in the reaction system,the method can achieve rapid detection of target sequences at room temperature（25°C）.Besides,the fluorescence signal showed a clear linear dependence（R²=0.9934）on the target strand concentration over the range from 0.01 to 10nmol/L.The regression equation was I_F=0.94x+1.45 with the limit of detection（LOD）estimated to be 9.8 pmol/L（3 times the signal-to-noise ratio calculation）.Investigation of sensitivity towards target RNA also displayed a good linear relationship（I_F=0.75x+1.18,R²=0.9810）,whose detection limit was 83 pmol/L.2.After investigating different surface connection methods,the more stable biotin-avidin system was selected as the immobilization technology for the capture probe H₁ on the magnetic ball.Based on CHA technology and magnetic separation,using DNA silver clusters as the non-covalently-labeled signal output,a non-enzymatic fluorescence detection method for viral nucleic acids has been developed.In this method,the synthesis of silver cluster was independent of the nucleic acid recognition process,and with the help of rapid enrichment and separation by magnetic balls,it can effectively avoid the false positive of fluorescence enhancement caused by non-specific hybridization,achieving low background signal.After optimization of the concentration and ration of sequences and reaction time of the CHA system,when the concentration of H₁ and H₂ was 3μM and 3.5μM,and reaction time was 3 h,the fluorescence signal and the target sequence showed a good linear correlation in the concentration range of0.01-10 nmol/L（y=766.3x+30.9,R²=0.9664）,with a detection limit of 0.82 nmol/L.Besides,this method showed good selectivity and repeatability in the control experiment with the base-mismatched sequence and the recovery test of serum-based matrix.3.During the study of nucleic acid detection method based on magnetic separation and CHA,it was found that the biotin-avidin coupling system had a significant quenching effect on the fluorescence of silver clusters（up to 90%）,and related verification studies were carried out.Firstly,we investigated the effect of the separation distance on the fluorescence intensity of the silver cluster by adjusting the number of T bases at the 3’-end of the biotin-labeled H₁（from 3T to 20T）.It was found that the quenching degree was the lowest（approximately 60%）when the separation distance was 7T.Furthermore,four more different silver clusters synthesized template sequences were chosen to verify that the system had fluorescence quenching effect on the all synthesized DNA silver clusters.