Fluorescence Quantitative PCR For Detection Of PAEs

Phthalate esters (PAEs) are plastic plasticizer, is widely used in the plastics industry, commonly found in soil, sediment, water, biological, air and atmospheric dust and other materials in environmental samples. Detection of phthalate esters are gas chromatography, liquid chromatography, gas chromatography-mass spectrometry (GC-MS), the fluorescence method. By chromatography is not only expensive and time-consuming, cumbersome and lengthy analysis process.In recent years, with the development of biotechnology, resulting in many biological detection methods for detect phthalates. Fluorescence quantitative PCR based on nucleotide colloidal dye have emerged in recent years, is a kind of new method with high-sensitivity.PAEs as estrogen receptor ligands can be combined with estrogen receptors and induce estrogen receptor-mediated gene expression. In this paper, exonuclease protection-quantitative PCR analysis method was established for the detection of phthalates.Based on the traditional digested protection, with S1 nuclease digestion after complete resection of ExoⅢleft a single strand of DNA can not be let free expansion, combined with SYBR Green I quantitative PCR technology for detection. The establishment of the exonuclease protection-RT-PCR method was applied to the water content of the test samples PAEs. In this study, GC-MS method of phthalate esters was compared with the new method. Experimental results are as follows:1. Expose carp buy from market to a certain concentration of phthalate in domesticated month later, taking receptor used in the experiment, found by electrophoresis after phthalate exposure over the fluorescence intensity was stronger than a group not exposed to carp. This proves that there are external environmental hormones phthalate, it can stimulate carp receptors to produce more.2. Using SPR to qualitative identification receptor-ligand complex results, when the phthalate standard solution concentration of 10-9～1g/L range, the concentration of phthalate ligand and receptor-ligand complexes SPR response to a linear relationship between intensity, indicating that the receptor-ligand complex with a good degree.3. Conventional PCR and quantitative PCR experiments were used. We got the relationship between phthalate's concentration and the Ct value:Ct=-1.2361og (phthalate's concentrations) +12.375, R2=0.9956. The results showed that when phthalate's concentration is in 10-8-10-2g/L, the linear relationship significant. When further reduce phthalate concentration, negative test results to determine the quantitative PCR experiments on phthalate minimum detection limit is 10-8g/L.4. This study also compared the quantitative PCR and GC-MS method, when using GC-MS methods for detection, the detection limit is 10-5 g/L. It can be seen, this new method of experimental detection limit lower than the GC-MS.5. In this study, quantitative PCR method phthalate was applied to the detection of environmental samples, environmental samples to determine the levels of phthalate, the results show that the method is accurate and feasible.In summary, this study successfully established a phthalate of SYBR Green I quantitative PCR monitoring methods, the method has low detection limit, high sensitivity, good accuracy, strong anti-interference, and is easy to use, it can be used as a new rapid and accurate monitoring of the environment in trace phthalate method.