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灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立

吕新 刘兰英 李玥仁

吕新,刘兰英,李玥仁. 灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立 [J]. 福建农业学报,2023,38(8):983−988 doi: 10.19303/j.issn.1008-0384.2023.08.013
引用本文: 吕新,刘兰英,李玥仁. 灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立 [J]. 福建农业学报,2023,38(8):983−988 doi: 10.19303/j.issn.1008-0384.2023.08.013
LV X, LIU L Y, LI Y R. SYBR Green I RT-PCR Assay for Quantitative Detection of Salmonella in Irrigation Water [J]. Fujian Journal of Agricultural Sciences,2023,38(8):983−988 doi: 10.19303/j.issn.1008-0384.2023.08.013
Citation: LV X, LIU L Y, LI Y R. SYBR Green I RT-PCR Assay for Quantitative Detection of Salmonella in Irrigation Water [J]. Fujian Journal of Agricultural Sciences,2023,38(8):983−988 doi: 10.19303/j.issn.1008-0384.2023.08.013

灌溉水源中沙门氏菌的SYBR Green I实时荧光定量PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2023.08.013
基金项目: 福建省科技计划公益类专项(2020R1022005);福建省农业科学院科技创新团队建设项目(CXTD2021011-3);福建省高质量发展超越“5511”协同创新工程项目(XTCXGC2021020)
详细信息
    作者简介:

    吕新(1980 —),男,硕士,助理研究员,主要从事农产品质量安全研究,E-mail:lux_ing@126.com

    通讯作者:

    李玥仁(1966 —),男,博士,研究员,主要从事农产品质量安全研究,E-mail:yuerenli@yeah.net

  • 中图分类号: S19

SYBR Green I RT-PCR Assay for Quantitative Detection of Salmonella in Irrigation Water

  • 摘要:   目的  建立基于SYBR Green I嵌合染料法的灌溉水源中沙门氏菌实时荧光定量PCR(Quantitative Real-time PCR, qPCR)检测方法。  方法  以沙门氏菌侵袭蛋白A (invA)基因为靶基因,设计1对特异性qPCR检测引物,在对qPCR反应体系和反应条件进行优化后,通过特异性实验和灵敏度实验对qPCR方法进行验证,并制作灌溉水源中沙门氏菌qPCR定量标准曲线,最后与国标方法对比检测灌溉水源中沙门氏菌污染来验证该方法的准确性。  结果  经过优化的qPCR检测方法灵敏度检测结果显示最低检测限量为1×10−1 pg·μL−1,特异性检测结果显示具有良好的特异性,干扰菌株对本qPCR检测方法无影响,所制作的qPCR扩增标准曲线在2×100~2×105 cfu·mL−1有较好的线性关系,相关系数为0.999 6,能对沙门氏菌进行准确的定量分析。该方法在与国标方法对比检测灌溉水源中沙门氏菌污染时具有同等准确性。  结论  本研究建立的基于SYBR Green I嵌合荧光法的实时荧光定量PCR检测方法,可以满足灌溉水源中沙门氏菌的定量检测要求。
  • 图  1  qPCR方法检测沙门氏菌

    A:qPCR荧光扩增曲线;B:qPCR溶解曲线。

    Figure  1.  Salmonella detection by newly developed qPCR assay

    A: qPCR amplification curve; B: qPCR melting curve.

    图  2  qPCR方法检测沙门氏菌的特异性

    Figure  2.  Specificity of qPCR assay in salmonella detection

    图  3  qPCR方法检测沙门氏菌的灵敏度

    Figure  3.  Sensitivity of qPCR assay in salmonella detection

    图  4  不同浓度沙门氏菌qPCR扩增曲线

    Figure  4.  qPCR amplification curve of detecting Salmonella at different concentrations

    图  5  沙门氏菌qPCR标准曲线

    Figure  5.  Standard curves of qPCR for salmonella detection

    图  6  qPCR扩增检测灌溉水源样品

    Figure  6.  qPCR quantification curves of detecting Salmonella in irrigation water

    表  1  供试菌株

    Table  1.   Salmonella strains applied for experimentation

    序号
    No.
    菌株名称
    Strains name
    拉丁学名
    Scientific name of strains
    菌株编号
    Strains number
    菌株来源
    Source of strains
    1鼠伤寒沙门氏菌Salmonella typhimurium1.1174CGMCC
    2金黄色葡萄球菌Staphylococcus aureus25923ATCC
    3大肠埃希氏菌Escherichia coli25922ATCC
    4阿氏肠杆菌Enterobacter asburiae1.876CGMCC
    5大肠埃希氏菌O157:H7Escherichia coli O157:H710907CICC
    6单核细胞增生李斯特氏菌Listeria monocytogenes1.9136CGMCC
    7英诺克李斯特氏菌Listeria innocua1.2990CGMCC
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-02-10
  • 修回日期:  2023-05-22
  • 网络出版日期:  2023-08-16
  • 刊出日期:  2023-08-28

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