Bio-barcode Immunoassay For Multi-reisdue Detection Of Organophosphorus Pesticides Based On Droplet Digital Polymerase Chain Reaction

The quality and safety of agricultural products is related to national health and social stability.Using pesticides illegally poses a great threat to the environment and even human health.In order to meet the requirements of pesticide residue standards,it is necessary to establish a high-sensitivity and high-accuracy detection method.The bio-barcode immunoassay detect the pesticides based on nucleotide chain signal amplification technology and magnetic nanoparticles separation technology.As an absolute quantitative detection technology,droplet digital PCR?ddPCR?is widely used in medicine,transgenic detection,microorganism detection,etc.Until now there is no report on pesticide detection by ddPCR.The DNA strands were collected from the immune competition reaction,which was detected by ddPCR.The main research contents and results are as follows:Preparing gold nonaparticle probes and magnetic nanoparticle probes,designing a set of highly specific DNA templates,primers and probes for PCR amplification,and optimizing the annealing temperature,upstream and downstream primers concentration and probes concentration for PCR amplification.The working concentration of gold nanoparticle probes and magnetic nanoparticle probes and methanol content were optimized.Under the optimal conditions,the detection limit of the triazophos(IC₁₀)was 0.002 ng mL^–1 and the IC₅₀ was 0.197 ng mL^–1.The linear range is 0.01-20 ng mL^–1.Spike recovery tests were carried out using apple,rice,cabbage and cucumber samples to verify the feasibility of the method.The recovery and relative standard deviations of the technique ranged from 76.9-94.4%and 10.8-19.9%,respectively.It was found that the linear between the proposed method and LC-MS/MS with respect to the recovery concentration was good,and the reliability of the proposed method was also proved.The the analytical method can be used for the limited detection of triazophos pesticide residues in agricultural products.A bio-barcode immunoassay based on ddPCR was established to detect triazophos,parathion and chlorpyrifos simultaneously.Two other DNA strands,upstream and downstream primers and probes were designed in this chapter.The annealing temperature,upstream and downstream primers and probes concentration of the two sets of deoxyribonucleic acid sequence during PCR amplification were optimized.The amounts of required parathion and chlorpyrifos deoxyribonucleic acid and mAbs were optimized.Experiments on the specificity between the three antibodies were made and the specificity was found to be good.The working concentrations of gold nanoparticle probes and magnetic nanoparticle probes were optimized.Under the optimal conditions,the detection limits of the triazophos,parathion and chlorpyrifos(IC₁₀)were 0.22 ng mL^-1,0.45 ng mL^-1 and 4.49 ng mL^-1,respectively.Spike recovery tests were carried out using apple,cucumber,cabbage and pear samples to verify the feasibility of the method.The recovery and relative standard deviations of the technique ranged from75.5–98.9%and 8.3–16.7%,respectively.The linear between the proposed method and LC-MS/MS with respect to the recovery concentration in the pear matrix was found to be good,which proved the accuracy and reliability of the bio-barcode immunoassay based on ddPCR.The bio-barcode immunoassay based on droplet digital PCR established in this study can detect triazophos,parathion and chlorpyrifos simultaneously.It also lays the foundation for high-throughput detection of more pesticides.