Research On Rapid Diagnosis Of Avian Influenza Based On AC Electrokinetic Effect And Impedance Immunosensing

Avian influenza(AI)is a respiratory or even systemic infectious disease caused by influenza A virus of the Orthomyxoviridae family.Among them,the highly pathogenic avian influenza has a sudden outbreak,a high mortality rate and a wide spread Features.At present,the Office International Des Epizooties(OIE)has designated Avian Influenza as a Class A infectious disease,and China has also classified it as a Class 1 animal infectious disease.Avian influenza is a zoonotic disease.Its outbreak not only causes serious economic losses to the poultry industry,but also poses a huge threat to public health security.At present,China takes avian influenza prevention and control as an important task.The prevention and control of avian influenza is a long-term and arduous task.The control of avian influenza not only requires the isolation of patients,but also the early detection of animal epidemics,so as to cut off the transmission of animals in a timely manner.At present,the main biological detection and diagnosis techniques for avian influenza viruses include isolation and detection of avian influenza viruses,such as HA-HI,ELISA,PCR and immunocolloidal gold.Virus detection and isolation are relatively accurate,but the experimental steps are tedious and time-consuming.The ha-hi test was sensitive,its specificity was low.ELISA is simple and suitable for the detection of large quantities of samples.but it needs to be repeated Washing is time-consuming and has limited detection sensitivity;although the PCR method can directly detect viral genes and has high detection sensitivity,the detection cost and technical requirements are relatively high.It is only suitable for laboratory diagnosis and is not suitable for rapid on-site diagnosis;for colloidal gold As for the detection technology,many products have been developed at present,but the sensitivity is a bit low,the product quality isinconsistent,and it can only conduct qualitative detection analysis,and can only be used for auxiliary detection of epidemic diseases.Therefore,aiming at the limitations of the current detection methods of avian influenza,it is of great significance to explore an efficient,specific,accurate and simple detection method for the timely prevention and control of avian influenza.The existing serological detection methods are mainly based on the reaction of antigen and antibody.At present,the quality of domestic avian influenza virus antibody products is uneven.There are relatively mature commercial antibodies abroad,but the price is very expensive.Therefore,the preparation of monoclonal antibodies of avianinfluenza virus with high valence,strong specificity and low cost can provide effective reagents for the detection,prevention and control of avian influenza.Fast and low-cost immunoassays are of great significance to global health.Traditional immunoassay methods cannot be completed quickly on site due to problems such as complicated processes.The method of enriching proteins with AC dynamics and based on impedance immunosensing is easy to operate,which can reduce the detection time of traditional ELISA from several hours to 1 minute,and also has a very low detection limit.This technology mainly uses AC electrokinetic effect to speed up the specific binding of antigen and antibody on the surface of the chip.At this time,the impedance immunosensor can monitor the change of the thickness of the electric double layer caused by the binding of antigen and antibody in real time to determine the positive result.This method has the characteristics of low cost,high sensitivity and wide detection range and is expected to be used for point-of-care testing(POCT)in clinical diseases.At present,there are foreign literature reports that the technology is used for the detection of bisphenol A(BPA),Zika virus RNA,Gram-negative bacteria,human D-Dimer,etc.,and all have achieved ideal experimental results.In the early stage,our laboratory has successfully applied this technology to the detection of avian Newcastle disease virus and Brucellosis antibody.The technology has not been reported in the detection of bird flu.In this study,a prokaryotic expression system was used to prepare H9N2 AIV hemagglutinin(HA)protein.The HA protein was used as an antigen in mice,and the mouse spleen cells with the required antibody titer were fused with myeloma(SP2/0)cells to produce monoclonal antibody.Furthermore,the HA antigen and antibody are used as raw materials for avian influenza diagnostic reagents,and the prepared HA antibody is added to the surface of the microelectrode chip after ozone treatment.(-OH)to increase the hydrophilicity of the chip electrode surface,which is conducive to the fixed binding of the antibody.In addition,the surface of the chip can be observed on the nano-scale uneven surface,which also increases the area of the antibody coated on the surface of the chip electrode.Then,the unbound sites are blocked to reduce non-specific binding,and a microelectrode chip for avian influenza antigen detection is prepared.The chip is connected to an impedance meter to monitor the immune response on the surface of the chip,and a preliminary establishment Avian influenza antigen detection method combining effect and impedance immunosensor,and evaluating the method,provides a new detection technology for on-site diagnosis of avian influenza.The main contents of this paper are as follows:(1)In order to achieve efficient expression of H9N2 AIV HA protein in prokaryotic expression system,the codon was optimized according to the A/chicken/Gansu/2/99(H9N2)HA(ID = EF070733.1)gene sequence released by GenBank,Chemically synthesize the entire HA gene,and design the restriction sites NcoI and XhoI at the 5 'and3' segments of the HA gene,and use DNA Ligation Kit to connect the HA gene to the pET28a(+)plasmid vector to construct the recombinant plasmid pET28a(+)-HA.Then pET28a(+)-HA was transformed into E.coli BL21(DE3),and the conditions(inducing temperature,inducing time,concentration of inducer)in the expression were optimized to express a large amount of HA protein.HA recombinant protein using a nickel column to Purification,the purified HA recombinant protein was subjected to preliminary purity analysis by polyacrylamide gel electrophoresis(SDS-PAGE);recombinant protein was purified by bisquinoline formic acid(BCA)method Determination of concentration;specific identification by Western blotting(Western-blot).(2)Using the method of long-range immunization,immunize BALB/c mice with the H9N2 AIV HA protein prepared above,take the mouse serum for antibody titer determination,and take the mouse spleen cells and antibody titers that meet the requirements of cell fusion.Myeloma cells(SP2/0)were fused with PEG1500;an indirect enzyme-linked immunosorbent assay(ELISA)for screening high-efficiency monoclonal antibodies was established,and hybridoma cells were cloned using the limiting dilution method and screened out.Monoclonal hybridoma cells that secrete antibodies stably.After expanding the cell line,appropriate cells are injected into the abdominal cavity of mice to prepare ascites antibodies.The ascites are collected and purified using Protein A column.The biological characteristics of the purified antibodies(Purity,concentration,potency,specificity).(3)Establish and optimize the AC dynamic impedance sensing detection method and apply it to the detection of avian influenza virus.First,optimize the cleaning conditions of the microelectrode on the surface of the chip,and then ozonize the chip to make the electrode surface with hydrophilic-OH and other groups to facilitate the immobilization of the antibody.Then,the purified anti-H9N2 AIV HA monoclonal antibody package Be on the surface of the chip and optimize the concentration and time of the antibody-modified chip,then optimize the blocking time,connect the chip to the impedance meter to detect avian influenza antigens,and optimize the voltage and current parameters of theimpedance meter to obtain the most suitable for detecting poultry Conditions for influenza antigens.Finally,the methods were evaluated in terms of sensitivity,specificity and repeatability.Results:(1)The genetically engineered strain pET28a(+)-HA/BL21(DE3)was successfully constructed,and the optimal induction conditions of the genetically engineered bacteria were selected.When the induction temperature was 30? and the IPTG concentration was0.8mmol/L,the induction was conducted for 6 hours.,The largest amount of HA protein;the purity of HA protein purified by nickel column affinity chromatography is high,reaching electrophoretic purity.In addition,Western blot results show that a protein band that matches the expected size appears at 63 KD,indicating the successful realization of HA Protein is highly expressed.(2)In the process of preparing H9N2 AIV HA monoclonal antibody,a total of two cell fusion experiments were performed,and the two fusion rates were 86.7% and 94.4%,respectively.The positive rates of initial tests were 33.5% and 86.75%,respectively.After repeated subcloning of the fused hybridoma cells three times,a total of 3 monoclonal cell lines capable of stably secreting anti-AIV HA antibodies were obtained,which were named2D10D10,1F10E7,and 5A10C3,1F10E7,5A10C3 are all IgG1 antibodies;the three hybridoma cell strains can secrete highly potent antibodies after repeated freezing,resuscitation and multiple passages.1F10E7 cells were injected into the abdominal cavity of mice to induce ascites antibody.Ascites were extracted and purified by protein A column.After purification,the purified antibody was identified by sds-page electrophoresis,and the purity of the purified antibody was reached by electrophoresis.After the concentration was determined by BCA method,the concentration could reach2.518mg/mL,Western-blot identification of the prepared HA monoclonal antibody confirmed that it can specifically bind to H9N2 AIV HA antigen and H9N2 standard virus,but does not react with NDV F protein,indicating that the prepared HA monoclonal antibody has better specificity.(3)In the process of establishing and optimizing the detection method of avian influenza antigen,the best chip cleaning method was determined as isopropyl alcohol ultrasonic cleaning for 10 min,absolute ethanol ultrasonic cleaning for 10 min,ultrapure water ultrasonic cleaning for 10 min;optimal antibody modification The chip conditions are: adding 10?g/mL HA monoclonal antibody and incubating in a 37? incubator for 3hours;blocking solution 1% super Block for 30 minutes at room temperature;the best voltage and current settings of the impedance meter are 100 mV and 100 kHZ.Coated HA monoclonal antibody was used to detect 0.1,1.0,10,100,1000ng/mL recombinant HA antigen dilution,the results showed that the method used to detect avian influenza antigen can detect the HA antigen of at least 10ng/mL,at this time detection The results are more stable.The chip was coated with HA monoclonal antibody to detect H9N2 negative positive poultry throat swab samples identified by fluorescent reverse transcription PCR(RT-PCR).A total of 12 positive samples(3 for each sample)and 6 negative samples were tested.In addition,4 samples of Newcastle disease virus allantoic fluid were tested,of which 3 samples were tested in parallel.Compared with the fluorescent RT-PCR detection method,the detection sensitivity(positive detection rate)can reach 97.2%,and the detection specificity(detection negative rate)can reach 96.7%,and the detection method does not cross with other common avian virus samples In response,the test results indicate that the test method has high specificity and repeatability.This method is expected to become a portable diagnostic system.The particle control chip coated with HA monoclonal antibody is treated with a protective agent and then stored in vacuum.Only a impedance device that is slightly larger than a mobile phone can be connected to the site during testing1 Test in minutes and report test results immediately.Conclusion:In this study,the H9N2 AIV HA genetically engineered strain pET28a(+)-HA/BL21(DE3)was constructed,and the optimal expression conditions of HA protein were screened to achieve large-scale expression of H9N2 HA protein in E.coli;As an immunogen for immunizing mice,after cell fusion and subcloning screening,three monoclonal cell lines that can stably secrete anti-H9N2 AIV HA antibodies were screened;Prophase preparation of antigen and antibody can be used to set up a communication galvanic effect combined with impedance sensor of rapid diagnostic methods of avian inflienza,after the optimization of the method and evaluation,proved that the method has high sensitivity and specificity,and good repeatability.It can be used for rapid detection of avian influenza,and provides a new detection technology for timely prevention and control of avian influenza.