Research On The Application Of Detection Of Immunoassay And Gene Biosensor During Agricultural Waste Composting

Composting is an effective disposal method for resource utilization of agricultural waste. Pesticide residue in agricultural waste affects the process of composting, and causes secondary pollution after compost products reused in soil. Enzyme-linked immunosorbent assay with high sensitivity and easy operation optimizes the composting process and controls the quality of the compost product, when applied to detection trace pesticide residue in the soil and composting process. Degradation of lignocellulose is the key content and rate-limiting step of agricultural waste composting. Detection of active enzyme involved in the degradation and its coding gene is good for composting process. DNA electrochemical sensor has the advantage of high sensitivity and specificity when used for detecting trace target functional genes in complex composting system, better reveals microbial feedback on composting environment, and provides accurate information for understanding and adjusting mechanism of degradation of lignocellulose in composting.This dissertation introduced competition detection mechanism to research of immunoassay and gene sensor applied in agricultural waste composting. A rapid and sensitive indirect competitive enzyme-linked immunosorbent assay was established for quantification of herbicide picloram in contaminated paddy soil and compost samples. A gold electrode and a screen printing electrode were modified by thiolated capture gene probe through self-assembling, the stable and orderly oligonucleotide monolayers based on the competitive hybridization for detection of target sequence of DNA from lignin degrading enzyme was developed. Many molecular biology methods was applied. This detection methods exhibit high sensitivity, specificity precision and accuracy.After immobilizing antigen, optimizing the concentrations of the goat-anti-rabbit IgG labeled by HRP and polyclonal rabbit-anti-picloram antibodies (PAb), antigen competed with picloram in tested solution for binding with antibody specifically, signal was detected and amplified by the goat-anti-rabbit IgG labeled by HRP, a rapid and sensitive indirect competitive ELISA was established for quantification of herbicide picloram in contaminated paddy soil and compost samples. The results showed that the optimal dilution ratio of PAb and the goat-anti-rabbit IgG were both 1:500(V/V). The detection limit for picloram by this method (LOD) was 5 ng·mL-1, and the linear detection range was 0.07 μg·mL-1～0.7μg·mL-1. In the recovery assay in paddy soil and compost extract samples, the recovery rate and coefficients of variation met the requirement of pesticide residue analysis. No significant interference from soil matrix was observed. This detection method exhibits high sensitivity and reproducibility, can achieve fast and convenient detection of bulk samples.A gold electrode was made by the use of glass and gold by burning and grinding and other physical methods. It was modified by thiolated capture gene probe through self-assembling, the stable and orderly oligonucleotide monolayers based on the competitive hybridization for detection of target sequence of synthetic DNA of lignin peroxidase(lip) gene of Phanerochaete chrysosporium was developed. Following hybridizations with competitively hybridized with the target nucleic acid and biotinylated response probe, streptavidin-horseradish peroxidase (HRP) conjugate was applied to the electrode. The electrochemical behavior was analyzed by cyclic voltammetry, electrochemical impedance spectroscopy, current-time curve and differential pulse voltammetry. The results show a good linear correlation between the current and the concentration of the target nucleic acid concentration in the range from 7.51×10-12 to 1.05×10-9 mol·L-1. The detection limit is 7.51×10-13 mol·L-1. The modified electrode exhibits high sensitivity, precision, stability and reproducibility.Gene probes and PCR primers was designed. Many molecular biology methods of gene extraction, polymerase chain reaction, agarose gel electrophoresis, gene purification, and nucleic acid hybridization was used for obtaining lip gene (265 bp) from Phanerochaete chrysosporium from pure culture and compost samples. The PCR products of lip gene (265 bp) was detected by the modified gold electrode with self-assembling oligonucleotide monolayers based on the competitive hybridization, The detection results were approaching by electrochemical biosensor and UV spectrophotometric measurement. The average recovery rate and coefficients of variation was 7.8% and 100.97% respectively. HRP-SA with high specificity and better signal amplification ability was screened as enzyme marker. This detection method exhibits high sensitivity, specificity, precision and accuracy.Pleurotus pulmonarius was inoculated in simulation agricultural waste composting culture fermentation system, laccase activity was measured and gene was extracted at different stages of culture, and dynamic change and development trend were comparative analyzed. Screen printing electrode (SPE) modified by thiolated capture gene probe through self-assembling, the electrochemical characterization showed the good stability of SPE under the applied voltage. The stable and orderly oligonucleotide monolayers based on the competitive hybridization for detection of target sequence of synthetic DNA of laccase gene of Pleurotus pulmonarius was developed. The results show a good linear correlation between the current and the concentration of the target nucleic acid concentration in the range from 0.25～17 ng mL-1. The detection limit is 9pg·mL-1. The modified electrode exhibits high sensitivity, precision, stability and reproducibility. The PCR products of lip gene (339 bp) was detected by the modified screen printing electrode with self-assembling oligonucleotide monolayers based on the competitive hybridization, The detection results were approaching by electrochemical biosensor and UV spectrophotometric measurement. The average recovery rate and coefficients of variation was 6.51% and 100.93% respectively. This detection method exhibits high sensitivity, specificity, precision and accuracy.