Colloidal Gold Nanoparticles-based Lateral Flow Immunoassay For Detection Of Bacillus Thuringiensis Crystal Protein Cry1C And Cry2A

Bacillus thuringiensis(Bt)genes Cry1C and Cry2A are widely used in crops to control Lepidoptera and Diptera insects.However,the biosafety of transgenic Bt crops and the traceability of exogenous Bt genes and exogenous Bt proteins are still controversial.In order to better control the biosafety issues and potential risks of Bt genetically modified crops,the detection and identification of Bt genetically modified crops is of great significance.In this project,colloidal gold nanoparticles(AuNPs)-based Lateral Flow Immunoassay(LFIA)for Bt protein Cry1C and Cry2A were developed and applied to the detection and identification of transgenic Bt crops.The specific conclusions are as follows:(1)This subject screened and obtained specific monoclonal antibodies against Cry1C(Cry1C;C3,C4,C6)through the monoclonal antibody preparation technology.Using immune colloidal gold technique,and the AuNPs-conjugated antibodies Cry1C-C4,Cry1C-C6 and goat anti-mouse IgG were used as capture antibodies,T-line detection antibody and C-line quality control antibody respectively,and the LFIA for Bt proteins Cry1C were developed.Under the optimal reaction conditions,the LFIA for Bt proteins Cry1C can complete the rapid detection of Cry1C within 10 minutes,and its visual detection limit is 15.6 ng/mL.Combined with mobile phone grayscale analysis software,the test strip has a detection range of 15.6-500 ng/mL(R~2=0.99)for Cry1C,and the detection limit of 10.42 ng/mL(2σ).The detection rate of Cry1C in Bt transgenic rice seeds by this method was 1%.Compared with commercial kits,the LFIA for Bt proteins Cry1C developed in this study has a better detection level and has a good commercial application prospect.(2)This subject screened and obtained specific monoclonal antibodies against Cry2A(Cry2A;A1,A2)through monoclonal antibody preparation technology.Using immune colloidal gold technique,and the AuNPs-conjugated antibodies Cry2A-A1,Cry2A-A2,goat anti-mouse IgG were used as capture antibodies,T Line detection antibody and C line quality control antibody respectively,and the LFIA for Bt proteins Cry2A were developed.Under the optimal reaction conditions,the LFIA for Bt proteins Cry2A can complete the detection of Cry2A within 15 minutes,and its visual detection limit is 625 ng/mL.Combined with mobile phone grayscale analysis software,the test strip has a detection range of 0.625-10.0μg/mL(R~2=0.98)for Cry2A,and the detection limit of 491 ng/mL(2σ).There is still a gap between the LFIA for Bt proteins Cry2A and commercial kits,and subsequent high-affinity paired antibodies can be rescreened for further research.