Study On A New Rapid Detection Method For Phage ?X174 Based On Flow Technology

In recent years,the government of our country has been constantly increasing the investment of science and technology in the biological industry,especially in the research work of the high pathogenic viruses,such as SARS,influenza A,and Ebola,from the outbreak since 2003.When performing experiments,researchers are easily exposed to infectious biomaterials,which can cause serious harm to their life and health.More and more attention has been paid to the detection and research of biosafety assessment,and concurrently,the performance and evaluation of personnel protective equipment is also particularly important.Therefore,the research on the new method of model virus measurement is actively and deeply conducted,and a more strict measurement standard is established to improve the accuracy of performance evaluation of personnel protective devices and to provide metrological support for monitoring pathogenic microorganisms in the environment.In this study,bacteriophage?X 174 was adopted as the research object.The reference materials of phage?X 174 was produced,the bacteriophage of?X 174 gpG and gpH protein genes were cloned and the expression vector was constructed,and the monoclonal antibodies against gpG and gpH proteins was prepared.The detection adopted flow cytometry as the basis to build the method of detecting bacteriophage?X 174.The specific experimental results are as follows:1.The cake-like reference material of bacteriophage?X 174 was prepared by freeze-drying technique.The prepared reference material of bacteriophage?X 174 had good homogeneity and stability,and can be stably preserved for four months.2.GpG and gpH genes has been successfully cloned and the expression vector was built up to induce and express the recombinant proteins gpG and gpH.3.With purified gpG and gpH proteins as antigens,monoclonal antibodies against gpG and gpH proteins were prepared.Ten hybridoma cell lines which can stably secrete anti-gpG and anti-gpH were obtained.4.For the first time,this study established a dual-fluorescent labeling method specific for viral nucleic acid and protein shells,which ensures that the detected virus is a live virus with infectivity.5.By optimizing the system parameters of flow cytometry and the fluorescence labeling method of bacteriophage?X 174,a method of detecting bacteriophage?X 174 by flow cytometry was established.The results of flow measurement showed a linear relationship,R~2=0.9998.There was a good linear relationship between the methods of flow detection and plaque counting,R~2=0.9985.The flow detection method established in this study had a high reproducibility and good stability.6.The method of detecting phage?X174 with virus counter was established by improving the labeling method of virus counter.The detection result generated by virus counter 3100presented a linear relationship,R~2=0.998.There was a good linear relationship between the virus counter 3100 and the plaque counting method,R~2=0.9904.The virus counter 3100 developed in this study had a high re-productivity and good stability.7.By optimizing the quantization imaging analysis on system parameters of the flow cytometry,a method of detecting bacteriophage?X 174 by a flow micro-imager was established.The detection result of the quantization imaging analysis of flow cytometry showed a linear relationship,R~2=0.988.There was a good linear relationship between the detection method of quantitation imaging analysis with flow cytometry and the plaque counting method,R~2=0.992.The flow micro-imager developed in this study had a high reproducibility and good stability.8.Compared with the method of plaque counting,the three flow detection methods established in this study have the advantages of short detection time,high accuracy,good re-productivity and others.The flow detection method can be promoted and applied to the detection of other pathogenic viruses.