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水环境中致病菌快速检测技术及应用研究进展

邓闵 李露 宋康

邓闵, 李露, 宋康. 水环境中致病菌快速检测技术及应用研究进展[J]. 环境科学研究, 2023, 36(10): 1835-1844. doi: 10.13198/j.issn.1001-6929.2023.08.11
引用本文: 邓闵, 李露, 宋康. 水环境中致病菌快速检测技术及应用研究进展[J]. 环境科学研究, 2023, 36(10): 1835-1844. doi: 10.13198/j.issn.1001-6929.2023.08.11
DENG Min, LI Lu, SONG Kang. Rapid Molecular Biology Detection and Risk Assessment of Waterborne Pathogens: A Review[J]. Research of Environmental Sciences, 2023, 36(10): 1835-1844. doi: 10.13198/j.issn.1001-6929.2023.08.11
Citation: DENG Min, LI Lu, SONG Kang. Rapid Molecular Biology Detection and Risk Assessment of Waterborne Pathogens: A Review[J]. Research of Environmental Sciences, 2023, 36(10): 1835-1844. doi: 10.13198/j.issn.1001-6929.2023.08.11

水环境中致病菌快速检测技术及应用研究进展

doi: 10.13198/j.issn.1001-6929.2023.08.11
基金项目: 国家自然科学基金项目(No.42222709)
详细信息
    作者简介:

    邓闵(1989-),男,安徽芜湖人,助理研究员,博士,主要从事水环境科学研究,dengm89@163.com

    通讯作者:

    宋康(1985-),男,江苏张家港人,研究员,博士,博导,主要从事水环境科学研究,sk@ihb.ac.cn

  • 中图分类号: X8;R123

Rapid Molecular Biology Detection and Risk Assessment of Waterborne Pathogens: A Review

Funds: National Natural Science Foundation of China (No.42222709)
  • 摘要: 介水传染病已经成为全球范围内的重要公共问题,对水环境中致病菌的适当监测和风险评估是预防水传播疫情的关键. 建立水环境中无需培养的快速致病菌检测方法并应用于QMRA(定量微生物风险评估)对于维护公众饮用水安全具有重要意义. 通过系统梳理发现,目前相关研究主要集中在分子生物学方法检测环境水体致病菌方法的建立方面,包括不同致病菌引物的设计,分析条件的优化,比较分子生物学方法与标准检测方法的一致性,以及提高分子生物学方法的灵敏度,降低检测用时和分析成本的探索等. 相对于传统培养方法,分子生物学方法在环境水体致病菌的检测方面具有灵敏度高、特异性好和耗时短的优势,此外,分子生物学方法还能够检测有活性但不可培养的菌株,区分致病毒株和非致病菌株,并在高通量检测致病菌方面具有不可替代的优势. 通过运用分子生物学技术快速定量致病菌浓度,并将其应用于QMRA中,可以提高对致病菌污染的预测能力. 最后,分子生物学技术可以更准确地识别不同种类致病菌的发生、传播程度、毒力,用于优化QMRA,但目前缺乏可用于评估的数据,未来建议在全国范围内设立重点监测站点,利用分子生物学技术收集长期的可在线访问的监测数据用于QMRA,结合人工智能模型,可以实现对区域范围的自动预警,从而提高保护公共健康的可能性.

     

  • 图  1  环境水体致病菌检测技术的发展历程

    Figure  1.  The developmental trajectory of pathogenic bacteria detection technologies in environmental water bodies

    图  2  定量微生物风险评估(QMRA)流程图

    Figure  2.  Flow diagram for quantitative microbial risk assessment (QMRA)

    表  1  不同致病菌检测方法及其检测限

    Table  1.   The detection methods for pathogens and their detection limits

    检测方法水体致病菌检测限检测时限数据来源
    荧光定量PCR技术 大肠杆菌、粪肠球菌(Enterococcus faecium) 27 cell/样本 文献[9]
    痢疾志贺菌 5 CFU/样本 <3 h 文献[11]
    肠球菌属(Enterococci spp.) 5~30 copies/样本 <3 h 文献[24]
    微阵列技术 分枝杆菌属(Mycobacterium spp.)、螺杆菌属(Helicobacter spp.)、变形杆菌属(Proteus spp.)、沙门氏菌属(Salmonella spp.)、大肠杆菌属、志贺(Shigella spp.)菌属、气单胞菌属(Aeromonas spp.)、弯曲杆菌属(Campylobacter spp.)、耶尔森菌属(Yersinia spp.)、霍乱弧菌(Vibrio cholerae)、拟态弧菌(V. mimicus)、河弧菌(V. fluvialis)、脆弱拟杆菌(Bacteroides fragilis)、长双歧杆菌(Bifidobacterium longum)、枸橼酸菌属(Citrobacter spp.)、产气荚膜梭菌(Clostridium perfringens)、粪肠球菌、单核李斯特菌(Listeria monocytogenes)、肺炎军团菌、绿脓杆菌(Pseudomonas aeruginosa)、类志贺邻单胞菌(Plesiomonas shigelloides)、无乳链球菌(Streptococcus agalactiae)、金黄色葡萄球菌(Staphylococcus aureus) 104 CFU/样本 交联2 h 文献[25]
    霍乱弧菌、副溶血性弧菌、溶藻弧菌(V. alginolytivus)、哈氏弧菌(V. harveyi)、创伤弧菌(V. vulnificus)、阴沟肠杆菌、嗜水气单胞菌 交联2 h 文献[26]
    沙门氏菌属、大肠杆菌、志贺菌属、小肠结肠炎耶尔森菌(Yersinia enterocolitca)、耶尔森菌属、弧菌属、肠杆菌科(Enterobacteriaceae)、霍乱弧菌、拟态弧菌、克雷伯菌属(Klebsiella spp.)、沙雷氏菌(Serratia spp.)、鼠疫耶尔森菌(Y. pestis)、肠杆菌属(Enterobacter spp.)、变形杆菌属、普罗威登斯菌属(Providencia spp.)、枸橼酸菌属 104基因组 交联4 h 文献[27]
    微流体qPCR技术 大肠杆菌属(Escherichia spp.)、侵袭性大肠杆菌(Enteroinvasive E. coli)、志贺菌属、沙门氏菌属、弯曲菌属(Campylobacter spp.)、梭菌属(Clostridium spp.)、肺炎军团菌、李斯特菌属(Listeria spp.)、弧菌属(Vibrio spp.) 100 cells/L 文献[28]
    绿脓杆菌、嗜水气单胞菌、克雷白氏肺炎杆菌(Klebsiella pneumoniae)、金黄色葡萄球菌 51 CFU/mL 文献[29]
    数字PCR技术 空肠弯曲菌、大肠杆菌、志贺菌属、沙门氏菌属 32 copies/(100 mL) <1 h 文献[30]
    多重PCR技术 嗜水气单胞菌、鲍氏志贺氏菌(S. flexneri)、小肠结肠炎耶尔森菌、鼠伤寒沙门氏菌(Salmonella typhimurium)、霍乱弧菌、副溶血性弧菌 100~102 CFU/样本 <12 h 文献[6]
    肠出血性大肠杆菌、志贺菌属、副溶血性弧菌、绿脓杆菌、沙门氏菌属 101~102 CFU/样本 6~8 h 文献[31]
    沙门氏菌属、肠出血性大肠杆菌O157(Eterohaemorrhagic E. coli O157)、绿脓杆菌、副溶血性弧菌、志贺菌属 101~102 CFU/样本 5~6 h 文献[32]
    沙门氏菌属、志贺菌属、绿脓杆菌、肠出血性大肠杆菌(Eterohaemorrhagic Escherichia)、副溶血性弧菌 101~102 CFU/样本 6~8 h 文献[33]
    LAMP技术 总大肠杆菌、绿脓杆菌、致病大肠杆菌O157:H7、鲍氏志贺氏菌、结核杆菌(Mycobacterium tuberculosis) 100 copies/样本 45 min 文献[7]
    副溶血性弧菌 1 cell/(100 mL) 1 h 文献[34]
    沙门氏菌属、空肠弯曲菌、志贺菌属、霍乱弧菌 10~100 copies/μL <20 min 文献[35]
    大肠杆菌、粪肠球菌 <10 CFU/样本 20 min 文献[36]
    肠道沙门氏菌(S. enterica)、空肠弯曲菌(Campylobacter jejuni)、肺炎军团菌、致病大肠杆菌O157:H7、霍乱弧菌 1 copy 19 min 文献[37]
    CRISPR基因编辑技术 致病大肠杆菌O157:H7 1 CFU/mL <55 min 文献[8]
    副溶血性弧菌 30 copies/样本 <50 min 文献[38]
    创伤弧菌 2 copies/样本 <40 min 文献[39]
    下载: 导出CSV

    表  2  不同致病菌检测技术优势与不足

    Table  2.   Advantages and limitations of different pathogen detection technologies

    检测方法优势不足
    荧光定量PCR技术 ● 已被美国环境保护局用于肠球菌检测标准方法
    ● 技术成熟度高
    ● 可区分活死菌
    ● qPCR检测设备价格昂贵
    ● 需要专业技术人员
    ● 依赖标准曲线
    ● 对环境水体中抑制物敏感
    微阵列技术 ● 检测通量极大,适合对环境高风险致病菌进行筛选 ● 检测限比qPCR高3~4个数量级,容易产生假阴性结果
    ● 检测成本高
    多重PCR技术 ● 检测通量大 ● 引物设计复杂
    ● 扩展性低,不同致病菌组合需要重新设计引物
    ● 检测时间比qPCR长
    ● 灵敏度比qPCR低1~2个数量级
    微流体qPCR技术 ● 所需样本量少
    ● 检测通量大
    ● 灵敏度比qPCR高
    ● 微流体qPCR设备昂贵
    ● 芯片成本高
    LAMP技术 ● 无需昂贵的热循环设备
    ● 直接检测水样
    ● 耗时短
    ● 需要多对引物,设计复杂
    ● 气溶胶污染易导致假阳性结果
    数字PCR技术 ● 不依赖标准曲线或外部校准品
    ● 耐受环境水体中抑制物
    ● 检测设备昂贵
    ● 数据分析依赖专业人员
    ● 检测成本高
    CRISPR基因编辑技术 ● 检测快速高效
    ● 灵敏度高
    ● 依赖参考基因组
    ● 依赖专业技术人员
    下载: 导出CSV
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  • 收稿日期:  2023-07-12
  • 修回日期:  2023-08-20
  • 网络出版日期:  2023-08-21

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