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水中抑制物对定量PCR与膜过滤法检测E.coli的影响

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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.
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  • 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。

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