Detection Of Porcine Delta Coronavirus Through Loop Mediated Isothermal Amplification Combined With Electrochemistry Or Enzyme Linked Immunosorbent Assay Based On Nano-enzyme

The outbreak and spread of the porcine delta coronavirus（PDCo V）has caused huge economic losses to pig breeding around the world,so it is of great significance to develop sensitive,convenient,low cost and high specificity detection methods.In this thesis,the detection of PDCo V at nucleic acid and protein levels were performed,including the loop-mediated isothermal amplification（LAMP）-electrochemical impedance detection platform.The detection of PDCo V with high sensitivity and specificity was realized.And through designing and preparing metal-organic framework（MOF）nano-enzyme,based on enzyme linked immunosorbent assay,the low cost,convenient and sensitive colorimetric detection of PDCo V was realized.The main research contents are as follows:1.Combining LAMP with high sensitivity,strong specificity and high amplification efficiency with the advantages of fast response,high sensitivity and excellent selectivity in electrochemical detection,the LAMP-electrochemical detection method of PDCo V was established.In this work,mercapto-modified primer（HS-FIP）was screened,designed and combined with PDCo V target sequence to initiate LAMP amplification.The LAMP-electrochemical impedance detection platform of PDCo V was constructed by linking the amplified double-stranded DNA with mercaptogroup on the surface of indium tin oxide glass deposited with Au NPs through gold mercapto bond reaction.Based on the difference of charge transfer resistances before and after LAMP amplification,the linear relationship betweenΔRet（ΔRet=R-R₀,where R and R₀were the charge transfer resistances of drop PDCo V plasmid amplification products and double distilled water as negative control respectively）and the concentration of PDCo V plasmid was found in the range of10²～10⁷copies/μL.The high sensitivity and specificity detection was realized.2.In order to lower the cost and realize portable detection,in this work nano-enzyme with good peroxidase activity instead of natural enzyme was designed and prepared.The colorimetric detection of PDCo V antigen was realized through enzyme linked immunosorbent assay.Firstly,the Fe-doped cerium MOF（Ce@Fe MOF）obtained by one-step process was calcined and its frame was partly carbonized to prepare Ce@Fe-C MOF with two-dimensional structure,bimetallic active center and good peroxidase activity.It was found that not only did the specific surface area of Ce@Fe-C MOF increase after calcination,but also the content of Ce³⁺ion,the peroxidase active center,increased.In addition,bimetallic active centers（Fe and Ce）had synergistic catalysis and higher peroxidase activity than monmetallic active center（Ce）.Further,the antibody of PDCo V was attached to the surface of Ce@Fe-C MOF by amidation reaction,and the colorimetric detection platform of PDCo V antigen through enzyme linked immunosorbent assay was constructed.Based on the good peroxidase activity of Ce@Fe-C MOF and the specific recognition of antigen and antibody,PDCo V antigen was quantitatively detected by the corresponding UV absorption intensity.The detection range was 10²～10⁷TCID50 m L^-1,and the detection limit was 33.33 TCID50 m L^-1.3.In order to further improve the catalytic efficiency and detection sensitivity of enzyme,Au NPs/Ce@Fe-C MOF nano-enzyme with double enzyme activity（glucose oxidase/peroxidase）was prepared by loading gold nanoparticles（Au NPs）with glucose oxidase activity on the surface of Ce@Fe-C MOF in the previous work.The cascade catalytic function of the double enzyme was used to improve the catalytic efficiency of the enzyme.In other words,Au NPs can catalyze glucose to produce gluconic acid and H₂O₂,Ce@Fe-C MOF can further catalyze H₂O₂to produce hydroxyl radicals,and then oxidized colorless 3,3’,5,5’-tetramethylbenzidine into blue oxidation state.At the same time,due to the excellent electrical conductivity of Au NPs,the electron transfer in Ce@Fe-C MOF was accelerated and the mass transfer resistance was reduced,thus improving the peroxidase activity of Au NPs/Ce@Fe-C MOF composites.The carboxyl group on the surface of Au NPs/Ce@Fe-C MOF was linked to the amino group on the surface of PDCo V antibody through amidation reaction.Based on the excellent enzyme activity of Au NPs/Ce@Fe-C MOF and the specific recognition of antigen and antibody,the colorimetric sensing platform with high sensitivity and strong specificity was constructed for PDCo V antigen analysis and detection.The linear range(10～10⁷TCID50 m L^-1)was further widened and the detection limit(3.33 TCID50 m L^-1)was lowered.