Separation And Purification Of Adenovirus And Influenza Virus From Water By Aqueous Two Phase Extraction Technology

In recent years,important acute infectious diseases occur frequently,especially virus infection（adenovirus,influenza virus,coronavirus,etc.）.It has a sudden onset,difficult diagnosis and treatment,high mortality,and poses a great threat to our people’s health,biological safety and social and economic stability.People or animals can be infected with pathogenic virus through drinking water,direct contact,etc.Virus pollution of water environment is one of the most serious pollution in the world.It is important to monitor adenovirus and influenza virus in water for preventing water borne diseases.Because the virus concentration in water is low and contains sand,protein,lipid and other impurities,it is usually necessary to concentrate and separate the virus before detection.Based on the characteristics of the aqueous two-phase extraction technology,which can selectively distribute the target substance and impurities into different phases and has good biocompatibility,a method for the separation and purification of adenovirus and influenza virus from environmental water samples was established,and the practical results of the established method and the conventional method were discussed.The first chapter mainly summarizes the current situation of virus pollution in water environment and its impact on human health,and summarizes the commonly used detection techniques and enrichment methods.At last,the background and content of this study are summarized.In the second chapter,a method for the isolation and purification of human adenovirus type 5（HAd V-5）by aqueous two-phase extraction was established.First,the virus in polyethylene glycol 6000（PEG 6000）was detected The results show that,due to the joint influence of the volume exclusion effect of PEG phase and the"salting out effect"of the bottom phase,it is easy to have lower interfacial tension between phases,so that HAd V-5has the best recovery effect at the junction of the top phase and the bottom phase after phase separation-"interphase phase",and the interphase volume is small,with less phase forming substances and high virus concentration,Therefore,phase to phase is determined as the best recovery location of HAd V-5.Then the concentration of the phase forming material,the p H of the system,the amount of sodium chloride added,the incubation temperature and time were optimized.The optimal experimental conditions were PEG 600024%（w/w）,sodium citrate 14%（w/w）,the top/bottom volume ratio（VR）was1.15,the p H was 8,without incubation,centrifugation for 3 min.the best recovery rate of HAd V-5 was 96.22%,and the total protein removal rate was71.01%.In the third chapter,the distribution of RNA virus（influenza virus H1N1）with envelope structure in PEG 6000 sodium citrate aqueous two-phase system was discussed.The results showed that the best recovery position was also in the phase.By optimizing the main influencing factors of the system,such as the concentration of phase forming substance,p H,incubation temperature and time,and the amount of sodium chloride,the best experimental condition was PEG 6000 20%（w/w）,sodium citrate 20%（w/w）,VR=0.84,p H 7,no incubation,centrifugation for 3 min.The best recovery of H1N1 was 94.87%,and the removal rate of total protein was 43.02%.Compared with HAd V-5,H1N1 needs lower concentration of PEG 6000 and higher concentration of Sodium Citrate under the optimal conditions.In the fourth chapter,the actual recovery effect of aqueous two-phase extraction technology in water samples was compared with membrane filtration elution method,ultrafiltration method and PEG precipitation method.The experiments were compared in two aspects:different virus content(1-5×10⁵TCID₅₀/m L)and different water volume（1-25 m L）.The results show that the recovery effect of the four methods is greatly affected by the virus concentration,and the higher the virus concentration is,the better the recovery effect is.The average recoveries of HAd V-5 and H1N1 were 0.24%and 3.85%,respectively.Ultrafiltration（average recoveries of HAd V-5 and H1N1 were 24.24%and 27.26%,respectively）and PEG precipitation（average recoveries of HAd V-5 and H1N1 were 20.02%and 34.82%,respectively）had similar virus recovery effect.However,due to the need of centrifugation,the two methods are suitable for the treatment of small water samples due to the limitation of experimental materials.Aqueous two-phase extraction technology has advantages in the recovery of HAd V-5 and H1N1in water samples,and the recovery effect of these two viruses is similar（the average recovery rates of HAd V-5 and H1N1 are 90.07%and 94.06%,respectively）.Compared with the conventional three virus concentration methods,the recovery rate of viruses extracted by aqueous two-phase extraction technology is higher,and it is stable in the amplification and reduction of the system,which is suitable for concentration and detection of unknown viruses in water samples viruses.The distribution of virus in aqueous two-phase system is first affected by the properties of phase forming substances,and then by the physical and chemical properties of virus itself,such as hydrophobicity,surface structure and isoelectric point.As a pretreatment method with great potential,aqueous two-phase extraction technology has the advantages of simple operation,economic and environmental protection,which can provide support for the monitoring of active viruses in the environment,and has great significance for the protection of public health safety.