高级搜索

水中抑制物对定量PCR与膜过滤法检测E.coli的影响

,  ,  ,  , 

downloadPDF

上一篇

下一篇

徐丽梅, 王晓昌, 周进宏, 吉铮, 张崇淼. 水中抑制物对定量PCR与膜过滤法检测E.coli的影响[J]. 环境工程学报, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
引用本文: 徐丽梅, 王晓昌, 周进宏, 吉铮, 张崇淼. 水中抑制物对定量PCR与膜过滤法检测E.coli的影响[J]. 环境工程学报, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
shu
Xu Limei, Wang Xiaochang, Zhou Jinhong, Ji Zheng, Zhang Chongmiao. Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method[J]. Chinese Journal of Environmental Engineering, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
Citation: Xu Limei, Wang Xiaochang, Zhou Jinhong, Ji Zheng, Zhang Chongmiao. Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method[J]. Chinese Journal of Environmental Engineering, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
shu

水中抑制物对定量PCR与膜过滤法检测E.coli的影响

  • 1.

    西安建筑科技大学环境与市政工程学院, 西北水资源与环境生态教育部重点实验室, 西安 710055

  • 基金项目:

    国家"水体污染控制与治理"科技重大专项(2013ZX07310-001)

  • 中图分类号: X832

Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method

  • 1.

    Key Laboratory of Northwest Water Resources, Environment and Ecology, Ministry of Education, School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China

  • Fund Project:

  • 摘要: 针对水中病原微生物的污染,选择大肠埃希氏菌E. coli作为病原示踪剂,以 E. coli染色体上β-葡萄糖醛酸酶uidA目的基因建立了SYBR Green实时荧光定量PCR的检测方法。该定量PCR方法灵敏度高,检测限可达104 CFU/L,线性关系良好,相关系数为R2=0.999。研究表明,定量PCR与菌液浓度呈显著正相关R2=0.935。通过人工投加腐殖酸、COD,研究了水中抑制物对定量PCR和膜过滤MF培养法的影响。结果显示,腐殖酸可使E. coli在培养皿上的菌落变小聚集,且显色不明显,当腐殖酸量为20 mg/L时,浓度为50 CFU/100 mL的E. coli完全受到抑制,没有菌落出现。腐殖酸对PCR扩增的抑制作用明显,当腐殖酸浓度增加到10 mg/L,定量PCR检测结果基因拷贝数减少约1 log,增加到20 mg/L定性PCR检测结果为阴性。
    Abstract: Escherichia coli (E. coli) was selected as the pathogenic tracer. Based on the uidA gene sequence of E. coli, the SYBR Green quantitative polymerase chain reaction (qPCR) method was established. The detected limitation of qPCR was 104 CFU/L and a good linear relationship was obtained with correlation coefficient (R2) of 0.999. The concentrations of E. coli detected by qPCR and membrane filtration (MF) method were presented a significant positive correlation with R2 of 0.935. Effect of inhibitors in water on detection of E. coli by qPCR and MF method was evaluated. Results showed that the colonies gathered together and became smaller, and the color of colonies was not apparent after adding humic acid. When concentrations of E. coli and humic acid were approximately 50 CFU/100 mL and 20 mg/L, respectively, colony growth was inhibited completely on culture plate. Inhibitory effect of humic acid on qPCR detection was apparent. As humic acid concentration increased to 10 mg/L, E. coli concentration reduced approximately for 1 log. While detected result was negative when humic acid concentration was 20 mg/L.
  • 加载中
  • [1] Scott T. M., Rose J. B., Jenkins T. M., et al. Microbial source tracking: Current methodology and future directions. Applied and Environmental Microbiology, 2002, 68(12): 5796-5803
    [2] Meays C. L., Broersma K., Nordin R., et al. Source tracking fecal bacteria in water: A critical review of current methods. Journal of Environmental Management, 2004, 73(1): 71-79
    [3] 谢润欣, 张崇淼, 王晓昌, 等. 城市污水中病原性大肠埃希氏菌毒力基因eaeA和rfbE的实时荧光定量PCR检测. 环境科学研究, 2012, 25(8): 922-926 Xie Runxin, Zhang Chongmiao, Wang Xiaochang, et al. Quantitative detection of virulence genes eaeA and rfbE of pathogenic Escherichia coli in municipal wastewater by real-time PCR. Research of Environmental Sciences, 2012, 25(8): 922-926 (in Chinese)
    [4] 安微, 张秀英, 李蕊, 等. 致病性大肠杆菌毒力因子和耐药性研究进展. 畜牧与兽医, 2013, 45(8): 106-109 Anwei, Zhang Xiuying, Li Rui, et al. The research progress of virulence factors and resistance of pathogenic Escherichia coli. Animal Husbandry & Veterinary Medicine, 2013, 45(8): 106-109 (in Chinese)
    [5] Bischoff C., Lüthy J., Altwegg M., et al. Rapid detection of diarrheagenic E. coli by real-time PCR. Journal of Microbiological Methods, 2005, 61(3): 335-341
    [6] International Health Regulations. Public health review of the enterohaemorrhagic Escherichia coli outbreak in germany. WHO, 2011
    [7] Lin C. K., Lin J. C. Development of PCR primers based on a fragment from randomly amplified polymorphic DNA for the detection of Escherichia coli O157: H7/NM. Molecular and Cellular Probes, 2007, 21(3): 182-189
    [8] Behets J., Declerck P., Delaedt Y., et al. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples. Water Research, 2007, 41(1): 118-126
    [9] Wilson I. G. Inhibition and facilitation of nucleic acid amplification. Applied Environmental and Microbiology, 1997, 63(10): 3741-3751
    [10] Bachoon D. S., Otero E., Hodson R. E. Effects of humic substances on fluorometric DNA quantification and DNA hybridization. Journal of Microbiological Methods, 2001, 47(1): 73-82
    [11] Takahashi H., Kimura B., Tanaka Y., et al. Real-time PCR and enrichment culture for sensitive detection and enumeration of Escherichia coli. Journal of Microbiological Methods, 2009, 79(1): 124-127
    [12] Maheux A. F., Picard F. J., Boissinot M., et al. Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Research, 2009, 43(12): 3019-3028
    [13] 张浩, 罗义, 周启星. 四环素类抗生素生态毒性研究进展. 农业环境科学学报, 2008, 27(2): 407-413 Zhang Hao, Luo Yi, Zhou Qixing. Research advancement of eco-toxicity of tetracycline antibiotics. Journal of Agro-Environment Science, 2008, 27(2): 407-413 (in Chinese)
    [14] Haugland R. A., Siefring S. C., Wymer L. J., et al. Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Research, 2005, 39(4): 559-568
    [15] Jaramillo Mdel C., Martínez-Duarte R., Hüttener M., et al. Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis. Biosensors and Bioelectronics, 2013, 43(1): 297-303
    [16] Gentry-Shields J., Wang A., Cory R. M., et al. Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation-emission matrix spectroscopy and PARAFAC. Water Research, 2013, 47(10): 3467-3476
    [17] Esau N., Matheson C. D., Gurney C. Assessing PCR inhibition from humic substances. The Open Enzyme Inhibition Journal, 2010, 3(10): 38-45
    [18] Schriewer A., Wehlmann A., Wuertz S. Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8. Journal of Microbiological Methods, 2011, 85(1): 16-21
    [19] Wang Meiyun, Olson B. H., Chang J. S. Improving PCR and qPCR detection of hydrogenase A (hydA) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin. Applied Microbiology and Biotechnology, 2007, 77(3): 645-656
  • 加载中
WeChat 点击查看大图
计量
  • 文章访问数:  2097
  • HTML全文浏览数:  1451
  • PDF下载数:  1142
  • 施引文献:  0
出版历程
  • 收稿日期:  2014-02-24
  • 刊出日期:  2015-02-07
徐丽梅, 王晓昌, 周进宏, 吉铮, 张崇淼. 水中抑制物对定量PCR与膜过滤法检测E.coli的影响[J]. 环境工程学报, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
引用本文: 徐丽梅, 王晓昌, 周进宏, 吉铮, 张崇淼. 水中抑制物对定量PCR与膜过滤法检测E.coli的影响[J]. 环境工程学报, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
shu
Xu Limei, Wang Xiaochang, Zhou Jinhong, Ji Zheng, Zhang Chongmiao. Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method[J]. Chinese Journal of Environmental Engineering, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
Citation: Xu Limei, Wang Xiaochang, Zhou Jinhong, Ji Zheng, Zhang Chongmiao. Effect of inhibitors in water on detection of Escherichia coli by quantitative PCR and membrane filtration method[J]. Chinese Journal of Environmental Engineering, 2015, 9(2): 609-614. doi: 10.12030/j.cjee.20150217
shu

水中抑制物对定量PCR与膜过滤法检测E.coli的影响

  • 1. 西安建筑科技大学环境与市政工程学院, 西北水资源与环境生态教育部重点实验室, 西安 710055
  • 收稿日期:  2014-02-24
基金项目:

国家"水体污染控制与治理"科技重大专项(2013ZX07310-001)

摘要: 针对水中病原微生物的污染,选择大肠埃希氏菌E. coli作为病原示踪剂,以 E. coli染色体上β-葡萄糖醛酸酶uidA目的基因建立了SYBR Green实时荧光定量PCR的检测方法。该定量PCR方法灵敏度高,检测限可达104 CFU/L,线性关系良好,相关系数为R2=0.999。研究表明,定量PCR与菌液浓度呈显著正相关R2=0.935。通过人工投加腐殖酸、COD,研究了水中抑制物对定量PCR和膜过滤MF培养法的影响。结果显示,腐殖酸可使E. coli在培养皿上的菌落变小聚集,且显色不明显,当腐殖酸量为20 mg/L时,浓度为50 CFU/100 mL的E. coli完全受到抑制,没有菌落出现。腐殖酸对PCR扩增的抑制作用明显,当腐殖酸浓度增加到10 mg/L,定量PCR检测结果基因拷贝数减少约1 log,增加到20 mg/L定性PCR检测结果为阴性。

English Abstract

    全文HTML

参考文献 (19)

返回顶部

目录

/

返回文章
返回

Copyright © 2019 中国科学院生态环境研究中心