Novel Methods For Molecule Detection Of Hepatitis Viruses Based On Magnetic Separation And Chemiluminescence

With the rapid development of nanotechnology,nanomaterials have been applied to the field of life science.Magnetic nanoparticles(magnetic,MNPs),as an important component of nanomaterials,have been widely used in the detection of various biological molecules.Chemiluminescence has the advantages of high security,high sensitivity and wide linear range,which provides a good platform for virus detection.In this paper,we have established several rapid,high-throughput,sensitive and practical techniques for the detection of hepatitis by combining the advantages of chemiluminescence and magnetic separation.Specific contents contain as following:1.Preparation of functionalized magnetic nanoparticles and characterizationIn order to improve the magnetic nanoparticles functionalized with the probes,The preparation of the traditional functionalized magnetic nanoparticles was modified before the molecular detection of hepatitis samples.Soft template method was applied into the preparation of Fe3O4 magnetic nanoparticles and subsequently coated with SiO2 and some of characterization methods are adopted.The results showed that Fe304 magnetic nanoparticles are round,and their average diameter is 500 nm,and shows uniform size with superparamagnetism whose saturation magnetization reached 1.7374 emu/g.Fe304 composite particles coated with SiO2 show obvious core-shell structure and their average particle size is 600 nm with size uniformity and good dispersion.The magnetic nanoparticles were applied to the extraction of nucleic acids from bacteria,and high extraction efficiency and purification were obtained.2.Extraction and amplification of nucleic acid molecules based on functionalized magnetic nanoparticlesCompared to extraction effency of genomic DNA from four different sources contained Escherichia coli,yeast,whole blood and serum,based on of Fe3O4@SiO2 nanoparticles as the carriers,the results showws that after the extraction of genomic DNA by gel electrophoresis test,extracted DNA band is uniform without towing phenomenon,and all the extracted DNA is complete,and no degradation is occurred.Finally,PCR amplification and restriction enzyme digestion experiments showed that the effect of the genomic DNA extracted for PCR amplification experiment is perfect.Enzyme analysis also indicated that genomic DNA extraction can be used for enzyme digestion experiment,so the genomic DNA from different sources can be extracted by magnetic separation experiments using magnetic nanoparticles the particles.The processing of The extraction of genomic DNA from magnetic beads is simple,fast,time-saving and labor-saving,without the pollution of chemical reagents,which is useful for large-scale genome nucleic acid extraction.This provides a basis for the determination of HBV molecular detection by the following high-throughput and multi-sample chemiluminescence.3.Establishment and optimization of PCR amplification detection method based on magnetic separation and chemiluminescenceThis chapter takes the synthetic biotin labeled hepatitis B nucleic acid fragment ssHBV simulation DNA as the target molecular,chemical luminescence hybridization detection method,ahepatitis B nucleic acid molecules were established.The results show that the good specificity of the method.To optimize the processing through a variety of experimental conditions on the detection system involved in,have a deeper understanding on the detection method,and obtains the optimized experimental conditions,is expected to improve the detection sensitivity of this method.The following optimized conditions:the best particle content is 100 g;the optimal concentration of SA MNPs was 0.1 mM-;the optimal concentration of amino modified probe into 2 M;the best hybr:idization temperature of 45 ?;the best time for hybridization was 30 min;the method in the low concentration range of 10-10000 copies/mL,a linear relationship the signal intensity,the sensitivity was 10 copies/mL.4.Establishment and optimization of the detection method for hepatitis B virus gene amplification based on magnetic separation and chemiluminescence combining with WGAThe whole blood by magnetic beads method to extract DNA template of hepatitis B,and HBV gene probe as an internal standard,were labeled with biotin containing hepatitis B blood samples,whole genome amplification,so as to establish a chemical marker of hepatitis B nucleic acid gene amplification luminescent hybrid detection method.The detection of hepatitis B virus by whole genome amplification technology is helpful to improve the detection sensitivity.The results showed that the specificity of the method was good,the hybridization efficiency of chemiluminescence is greatly improved,to optimize the experimental conditions and results are as follows:the optimum particle content is 80 g;the optimal concentration of SA MNPs was 0.1 mM;the optimal concentration of amino modified probe into 100 M;the optimal hybridization time was 40 min.5.Method of virus detection based on loop mediated isothermal amplification and chemiluminescenceAt present there are many methods used for clinical detection of the virus,such as polymerase chain reaction(PCR),real time fluorescence quantitative PCR(real-time qPCR),enzyme-linked immunosorbent assay(ELISA)and chemiluminescence(CL)detection etc..In these detection techniques,chemiluminescence detection based on nucleic acid hybridization has been widely used in the detection of hepatitis B virus DNA and hepatitis C virus RNA.However,the traditional chemiluminescence detection not only by polymerase chain reaction(PCR)to obtain the target DNA effectively,so as to be used for hybridization,also need long time thermal cycling process of complex and expensive instruments,these deficiencies make this method is not suitable for community hospitals or on-site monitoring in medical conditions-are relatively low.The loop mediated isothermal amplification(LAMP)technique allows nucleic acids to be amplified in a fast and highly specific manner under isothermal conditions,avoiding the long and complex thermal cycling of PCR and expensive experimental instruments.In this paper,a method based on reverse transcription loop mediated isothermal amplification(RT-LAMP)and chemiluminescence was used to detect RNA virus.Biotin-11-dUTP joined in LAMP amplification to PCR products with biotin labeled,after hybridization with specific probes,finally using ALP-AMPPD chemiluminescence reaction system to deteRMine if the samples contain viral nucleic acid,in order to achieve the purpose of pathogen detection.In this study,we optimized the conditions of LAMP amplification and probe hybridization,such as LAMP reaction temperature,hybridization time,hybridization temperature,probe concentration and so on.The reaction temperature is 65 ?and 61? on HBV and HCV gene amplification efficiency;probe concentration is 10 M,the hybridization time was 50 min for HBV and HCV probe;however,two gene probes the optimal hybridization temperature is slightly different,the optimum temperature of 45 ? hybrid HBV genes and HCV,the optimum temperature is the same to 45 ? hybridization probe.Finally,through the sensitivity test,we obtain that this method can detect 103 copies/mL of HCV-RNA.Compared with the traditional PCR based chemiluminescence detection,the detection time was shortened by nearly 1 h.This method is highly specific for nucleic acid detection based on LAMP amplification and nucleic acid hybridization.This method simplifies the detection process and makes it easier to automate,so it has a broad application prospect in clinic.6.Application of Functional Microsphere in Human Hepatitis B Virus Surface Antigen DetectionA novel and simple emulsifier-free emulsion polymerization technique was developed for preparation of mono-dispersed amino functionalized polymer microspheres with well defined diameters(about 400 nm).Various characterization methods demonstrated that the obtained amino microspheres had a uniform size and good dispersity which were confirmed by scanning electron microscope(SEM).Zeta potential and Fourier transform infrared spectrometer(FT-IR)demonstrated that amino groups have been successfully introduced to the microsphere surface.These functionalized microspheres have been shown to be efficient and controllable carriers capable of immobilizing and enriching monoclonal antibodies.Moreover,a newest chemiluminescent enzyme-linked immunoassay(ELISA)approach has been developed for human Hepatitis B virus surface antigen(HBsAg)detection.HBsAg was sandwiched between goat anti-HBsAg polyclonal antibody coated on microspheres and mouse anti-HBsAg antibody.Alkaline phosphatase(ALP)conjugated horse anti-mouse immunogloblin was used to bond with monoclonal antibody.Finally,chemiluminescent(CL)signals were recorded after adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)phenyl-1,2-dioxetane(AMPPD)which was used as a chemiluminescent substrate reagent of ALP.This novel chemiluminescent ELISA assay was proved to be of excellent specificity and high sensitivity when using ALP and AMPPD luminescence systems for specific HBsAg detection.