Establishment Of Methods For Detecting Norovirus Based On DNA Self-Assembly Biosensors

Norovirus,also known as norwalk virus,is a prototype of the genus norovirus in the family human calicivirus.It is a group of viral particles with similar morphology and slightly different antigenic.The clinical manifestations are vomiting,diarrhea and headache and other symptoms.Because of its rapid evolution and lack of appropriate animal culture model and in vitro cell system,so that there is no vaccine developed yet,and because of its low pathogenic dose and highly stable characteristics,increased its difficulties in prevention and treatment,can easily lead to large-scale group infection event.The food safety issues caused by norovirus are not only easy to cause international trade barriers,but also bring a great threat to people’s health,especially in the areas where resources are scarce,medical equipment is behind,professional health care workers are scarce and sanitation is worrying,it is more than the hardest hit by the norovirus.Traditional norovirus detection methods are mainly electron microscopy,immunology and reverse transcription PCR.Although these methods have certain effectiveness,there are still some shortcomings.Among them,the sensitivity of electron microscopy is low and visual interference is serious,immunological method is cumbersome and requires a lot of antigens and antibodies which need cryopreservation and professional operators,reverse transcription PCR is subject to a large number of PCR inhibitors in the sample,resulting in low amplification efficiency.Therefore,there is still a need to develop a simple,accurate and fast detection method for noroviruses.In this study,a highly conserved sequence between ORF1 and ORF2 was used as a detection target.Two kinds of signal amplification methods were designed based on nanotechnology and DNA self-assembly biosensors in the case of minimizing the need for enzymes and antigenic antibodies requiring special storage conditions,but also to ensure their sensitivity,with a view to lay the foundation for the development of simple and fast,low cost and on-site norovirus detection methods,the development of a unified detection standard and the development of norovirus vaccine.1.A new method of norovirus detection was established based on the self-assembly of DNA and nanomaterials.Norovirus was used as the detection object and the highly conserved sequence between ORF1 and ORF2 of the genome was used as the research target.DNA self-assembly technique was used to synthesize X-DNA as the capture probe to increase the target DNA binding rate.Psoralen was used to enhance the stability of the capture probe and verified by agarose gel electrophoresis.The magnetic nanoparticles were used to amplify the detection signal by the self-assembly technique of DNA and nanomaterials and a series of characterization methods were carried out,such as transmission electron microscopy,X-ray diffraction analysis,magnetic properties analysis and surface-enhanced raman scattering spectroscopy analysis.Then the reaction solution was added to QCM and the parameters were optimized.The optimal parameters were obtained:the concentration of capture probe was 1 μM,the hybridization temperature was 37 ℃,and the reaction time was 3 h.Under the optimum reaction condition,the quantitative detection of norovirus was realized by quartz crystal microbalance sensor.The results show that the linear detection range was 1 pM to 1 μM,the linear correlation coefficient was 0.9956,the detection limit was 0.15 pM and the detection of target DNA effective and real-time was realized.2.Establishment of a method for detecting norovirus based on the combination of DNA nanoclusters and microcantilever beam sensors.The single-stranded DNA was ligated with the viscous ends of X-DNA by DNA base complementary pairing principle to self-assemble as DNA nanoclusters.The self-assembly parameters such as salt ion concentration,temperature,time,pH and water were optimized.The highly conserved sequence between ORF1 and ORF2 was used as the research target and the microcantilever beam sensor was used for real-time detection of norovirus.The feasibility of the experiment was verified by the prepared gold nanoparticles simulated microcantilever sensor connected to the capture probe.The target DNA was used as a bridge to connect the DNA nanoclusters and the capture probe to realize the detection signal amplification.The results show that the linear range was 10 pM～1 μM and the linear correlation coefficient was 0.9909.The addition of DNA nanoclusters not only amplified the detection signal but also improved the relevance of the method to facilitate accurate quantitative analysis.3.This study initially established two methods for the detection of norovirus basing on nucleic acid probes,which laid a foundation for the development of simple,rapid and inexpensive on-site virus detection methods,the development of uniform detection standard and the development of norovirus vaccine,has a positive practical application value.