With the rapid development of nanotechnology, nano materials 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, such as nanoparticles. 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 include:
1. Preparation of functionalized magnetic nanoparticles and their application in nucleic acid extraction
In order to improve the magnetic nanoparticles functionalized with probe, in this paper the high sensitive detection of hepatitis B virus samples of functional magnetic nanoparticles before preparation was improved by MNPs prepared by soft template method has obvious core-shell structure, the average diameter is 500 nm, and the circular particles, uniform size, superparamagnetism, saturation magnetization of 1.7374 emu/g, prepared by SiO2 coated Fe3O4 composite particles, the average particle size is 700 nm, with good dispersion, particle size uniformity is round. The magnetic nanoparticles were applied to the extraction of nucleic acids from bacteria and whole blood samples, and good extraction results were obtained.
2. Extraction and amplification of nucleic acid molecules based on functionalized magnetic nanoparticles
Firstly, two kinds of hepatitis B nucleic acid extraction methods were compared and analyzed, and the results were analyzed. The results showed that although the amount of nucleic acid extracted from serum hepatitis virus was small, it could be used in PCR amplification, but it was less effective in whole genome amplification. At the same time, the amount of nucleic acid extracted from the whole blood is high, on the one hand, it can be used for PCR amplification, and can also be applied to the whole genome amplification technology. Whole genome amplification of whole blood hepatitis B nucleic acid DNA can also be used for PCR amplification. In this chapter, we also optimized the whole genome amplification condition of whole blood hepatitis B virus DNA, which laid the foundation for the high-throughput detection of hepatitis B virus detection by chemiluminescence.
3. Establishment and optimization of PCR amplification detection method based on magnetic separation and chemiluminescence
This chapter takes the synthetic biotin labeled hepatitis B nucleic acid fragment ssHBV simulation DNA as the target molecular, chemical luminescence hybridization detection method, a hepatitis 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 hybridization temperature of 45 C; 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. Detection of hepatitis C virus RT-PCR amplification based on magnetic separation and chemiluminescence
Hepatitis C is a kind of hepatitis caused by RNA virus, which is different from hepatitis B through the HBV DNA virus replication, its infectivity and concealment is strong, social harm is bigger. Based on the early detection of hepatitis B virus, we set up a method for detecting RNA virus nucleic acid based on magnetic separation and chemiluminescence signal amplification. We use HCV RNA as the research object, with the C RNA feature gene for object detection, extraction of virus nucleic acid in DNA magnetic beads method for RT-PCR amplification. The target sequence, through both hybridization after specific chemiluminescence detection.
5. Establishment and optimization of the detection method for hepatitis B virus gene amplification based on magnetic separation and chemiluminescence
The whole blood by magnetic beads method to extract DNA template of hepatitis B, and GADPH gene probe as an internal standard, were labeled with biotin containing hepatitis B HBV blood, 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 HBV 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.
6. Method of virus detection based on loop mediated isothermal amplification and chemiluminescence
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 63 C on HA and NA gene amplification efficiency; probe concentration is 10 M, the hybridization time was 50 min for HA and NA probe the best effect; however, two gene probes the optimal hybridization temperature is slightly different, the optimum temperature of 50 hybrid HBV genes, the optimum temperature is 55 hybridization probe for HCV. 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.
7. Application of Functional Microsphere in Human Hepatitis B Virus Surface Antigen Detection
A 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 immunnogloblin was used to bond with monoclonal antibody. Finally, chemiluminesent (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.