邻苯二甲酸二丁酯的荧光定量PCR检测新方法研究
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摘要
本课题研究目的旨在建立采用荧光定量PCR检测邻苯二甲酸二丁酯浓度的新方法。邻苯二甲酸二丁酯可以活化雌激素受体使之与包含特定序列的双链DNA结合,结合的DNA因受到保护可抵抗核酸外切酶ExoⅢ的消解而被保留,痕量的保留DNA可通过PCR扩增定量。根据该原理,首先从鲤鱼肝脏细胞中提取含雌激素受体的细胞溶质,再与不同浓度的邻苯二甲酸二丁酯结合使雌激素受体活化,形成配体-受体复合物。然后,以双链pUCl9质粒DNA作为模板,设计合成特异性引物序列,通过普通PCR扩增制备双链结合DNA,使双链结合DNA与配体-受体复合物反应结合后,用核酸外切酶ExoⅢ和S1核酸酶消解,去除未受到保护的DNA。利用消解后产物作为模板,进行荧光定量PCR扩增反应,从而建立Ct值与邻苯二甲酸二丁酯浓度的标准曲线。同时探索最优反应条件,并确定荧光定量PCR的最低检测限及其结果的重复性。将该方法用于检测实际环境样品中邻苯二甲酸二丁酯的含量,并将检测结果同高效液相色谱法进行比较,比较两种检测方法的不同。
实验结果表明,邻苯二甲酸二丁酯浓度在10-5 g/L至10-10g/L之间时,邻苯二甲酸二丁酯浓度与Ct值线性关系显著。当邻苯二甲酸二丁酯浓度为10-10g/L时,结果呈正相关性。在该方法中,邻苯二甲酸二丁酯的检测下限是8.439 pg/L。将荧光定量PCR技术用于检测水和土壤样品中的邻苯二甲酸二丁酯浓度,其检测结果分别为0.1058μg/L和0.1357 ug/g。
同时在高效液相色谱法与荧光定量PCR方法检测邻苯二甲酸二丁酯的比较中,发现荧光定量PCR具有反应灵敏、检出限低、重现性好、操作简便,可同时处理大批量样品等优点。因此荧光定量PCR可作为一种有效的痕量邻苯二甲酸二丁酯定量检测方法。
In this study, a novel method to detect Dibutyl phthalate (DBP) using fluorescence quantitative PCR (FQ-PCR) was developed.
In this study, when the concentrations of DNA probes were in the range of 109 to 103 copies/μl, the correlation between Ct values and DNA copies was linear. (R2=0.99713).The analysis of melting curve show that Tm value was consistent with the expected, and there was only one remarkable peak, so this standard curve could be used as quantitative standard curve.
Extracted Estrogen receptor from the fish liver cells, and incubated the solute with different concentrations of DBP to make it activation. The combination of estrogen receptor and DBP come into receptor-ligand complex. Then, amplified through conventional PCR, specific primers were designed and synthesized in company with pUC19 as template for preparation of double-stranded DNA with estrogen responsive elements. Hydroxyapatite (HAP) was used to purification the ligand-receptor complex, and the Result shows that it's efficiency. These ligand-receptor-DNA complexes were digested by EXOIII and S1 nuclease. S1 stop buffer was added to inactivate S1 nuclease. In this experiment, the relationship between the concentrations of DBP and Ct values were analyzed from the quantitative standard curve and the plot between DNA copies and concentrations of DBP. These Results show that there was linear relationship when DBP concentrations were between 10-5 g/L and 10-10 g/L. When DBP concentration was 10-10 g/L, the Result was positive. So the lowest detection limits of this method 8.439 pg/L for DBP. Furthermore, FQ-PCR was able to detect DBP concentration in water and soil sample. In this study DBP concentration in soil and water can be detected at 0.1357 ug/g and 0.1058μg/L.
In this study, SPR was used to detect and identification the quantization of combination of Estrogen receptor and DBP. In this research HPLC method that can be detected lowest DBP concentration at 0.0084μg/L and the linear equation of concentration area standard curves are y =0.00121x-0.05818 (R2=0.99855) and the concentration of DBP in soil samples and water samples are 2.7319 ug/g. and 0.2129μg/L. Compare with the lowest detection limit of HPLC method that can be detected. FQ-PCR could be detected through the concentration of DBP in samples lower than HPLC method.
实验结果表明,邻苯二甲酸二丁酯浓度在10-5 g/L至10-10g/L之间时,邻苯二甲酸二丁酯浓度与Ct值线性关系显著。当邻苯二甲酸二丁酯浓度为10-10g/L时,结果呈正相关性。在该方法中,邻苯二甲酸二丁酯的检测下限是8.439 pg/L。将荧光定量PCR技术用于检测水和土壤样品中的邻苯二甲酸二丁酯浓度,其检测结果分别为0.1058μg/L和0.1357 ug/g。
同时在高效液相色谱法与荧光定量PCR方法检测邻苯二甲酸二丁酯的比较中,发现荧光定量PCR具有反应灵敏、检出限低、重现性好、操作简便,可同时处理大批量样品等优点。因此荧光定量PCR可作为一种有效的痕量邻苯二甲酸二丁酯定量检测方法。
In this study, a novel method to detect Dibutyl phthalate (DBP) using fluorescence quantitative PCR (FQ-PCR) was developed.
In this study, when the concentrations of DNA probes were in the range of 109 to 103 copies/μl, the correlation between Ct values and DNA copies was linear. (R2=0.99713).The analysis of melting curve show that Tm value was consistent with the expected, and there was only one remarkable peak, so this standard curve could be used as quantitative standard curve.
Extracted Estrogen receptor from the fish liver cells, and incubated the solute with different concentrations of DBP to make it activation. The combination of estrogen receptor and DBP come into receptor-ligand complex. Then, amplified through conventional PCR, specific primers were designed and synthesized in company with pUC19 as template for preparation of double-stranded DNA with estrogen responsive elements. Hydroxyapatite (HAP) was used to purification the ligand-receptor complex, and the Result shows that it's efficiency. These ligand-receptor-DNA complexes were digested by EXOIII and S1 nuclease. S1 stop buffer was added to inactivate S1 nuclease. In this experiment, the relationship between the concentrations of DBP and Ct values were analyzed from the quantitative standard curve and the plot between DNA copies and concentrations of DBP. These Results show that there was linear relationship when DBP concentrations were between 10-5 g/L and 10-10 g/L. When DBP concentration was 10-10 g/L, the Result was positive. So the lowest detection limits of this method 8.439 pg/L for DBP. Furthermore, FQ-PCR was able to detect DBP concentration in water and soil sample. In this study DBP concentration in soil and water can be detected at 0.1357 ug/g and 0.1058μg/L.
In this study, SPR was used to detect and identification the quantization of combination of Estrogen receptor and DBP. In this research HPLC method that can be detected lowest DBP concentration at 0.0084μg/L and the linear equation of concentration area standard curves are y =0.00121x-0.05818 (R2=0.99855) and the concentration of DBP in soil samples and water samples are 2.7319 ug/g. and 0.2129μg/L. Compare with the lowest detection limit of HPLC method that can be detected. FQ-PCR could be detected through the concentration of DBP in samples lower than HPLC method.
引文
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[4]Ching, N. P. H., Jham, G. N., Subbarayan, C., Bowen, D. V., Smit, A. L. C., JR., Gross, C. E.,Hicks, R. G., Field, F. H., AND Nealon, T. F., JR. Gas chromatographic-massspectrometric detection of circulating plasticizers in surgical patients. Journal of Chromatogr.1981,222:171-177.
[5]Foster, P. M., Cattley, R. C., and Mylchreest, E. Effects of Di-n-butyl phthalate (Dibutyl Phthalates) on male reproductive development in the rat:Implications for human risk assessment. Toxicology science,2000,38(1 Suppl),97-99.
[6]Shanna, H. S., Katharina, M. M., Fan, L., Sara, L. S., Robin, L. K., Antonia, M. C., Catherine, S. M., J. Bruce, R., Christine, L. T., Shannon, S., J.Lynn, T., and The Study for Future Families Research Team. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environmental Health Perspectives,2005,113,1056-1061
[7]Main, K. M., Mortensen, G. K., Kaleva, M. M., Boisen, K. A., Damgaard, I. N., Chellakooty, M., Schmidt, I. M., Suomi, A. M., Virtanen, H. E., Petersen, D. V., Andersson, A. M., Toppari, J., and Skakkebaek, N. E. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environmental Health Perspectives 2006,114(2):270-276.
[8]Yunhui, Z., Xuezhi, J., and Bingheng, C. Reproductive and developmental toxicity in F1 Sprague-Dawley male rats exposed to di-n-butyl phthalate in utero and during lactation and determination of its NOAEL. Reproductive Toxicology,2004,18(5):669-76.
[9]Daniel, S. M. NTP Technical Report on Toxicity Studies of Dibutyl Phthalate National Toxicology Program. NIH Publication,2005,95-3353.
[10]Kevin, W. G., Janan, B. H., Delong, L., Duncan, G. W., Susan, B., Kamin; J. J., Susan, J, H., and Kim, B. Fetal Mouse Phthalate Exposure Shows that Gonocyte MuLtinucleation is Not Associated with Decreased TesticuLar Testosterone. Toxicology Science,2007, 97(2):491-503.
[11]Li, X. H. Ma, L. L. Liu, X. Fu, F. S. Cheng H. X. and Xu, X. B. Phthalate esters pollution in Urban soil of Beijing, Bulletin of Environmental Contamination and Toxicology,2006, 77:252-259.
[12]EPA.2000a. Designation of hazardous substances. U.S. Environmental Protection Agency. Code of Federal Regulations. http://www.access.gpo.gov/nara/cfr/waisidx_00/ 40cfr14_00.html. October 25,2000.
[13]DeLeon, I.R., Byrne, C.J., and Peuler, E.A, Trace organic and heavy metal pollutants in the Mississippi River. Chemosphere,1986,15(6):795-805.
[14]Sheldon, L.S., Hites, R.A.. Sources and movement of organic chemicals in the Delaware River. Environmental Science and Technology,1979,13(5):574-579.
[15]McFall, J.A., Antoine, S.R and DeLeon, I.R.. Base-neutral extractable organic pollutants in biota and sediments from Lake Pontchartrain. Chemosphere,1995.14(10):1561-1569.
[16]Michele, D. C., Alessia, P., Giampiero, S., Dario, C., Dino, M., and Angelo, C. Determination of phthalate esters in wine using solid-phase extraction and gas chromatography-mass spectrometry. Food Chemistry,2008,111(3); 771-777
[17]Prapatpong, P. and W. Kanchanamayoon. Determination of phthalate esters in drinking water using solid-phase extraction and gas chromatography. Journal Applied Science, 2010,10:1987-1990.
[18]Coldham, N. G., Dave, M., and Sauer, M. J. Analysis of di-n-butyl phthalate biotransformation in cattle by liquid chromatography/ion trap mass spectrometry/mass spectrometry. Journal of Mass Spectrophotometer,1998,33:803-810.
[19]Van Vliet, H.P.M. Bootsmann, Th.C. Frei, R.W. and Brinkman, U.A.Th. On-line trace enrichment in high-performance liquid chromatography using a pre-column. Journal of Chromatography A.1979,185;483-495.
[20]United States Environmental Protection Agency.Method 1996. Method8061A:Phthalate esters by gas chromatography with electron capture detection (GC/ECD). Washington, DC:U.S.
[21]Chee, K.K. Wong, M.K. and Lee, H.K. Microwave extraction of phthalate esters from marine sediment and soil. Chromatographia,1996,42(7-8):378-384
[22]United States Environmental Protection Agency.1986a. Method 8060:Phthalate esters. In:Test methods for evaluating solid waste.3rd ed. SW-846. Washington, DC:U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response.
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