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沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用

吕新 刘兰英 陈丽华 李玥仁

吕新,刘兰英,陈丽华,等. 沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用 [J]. 福建农业学报,2021,36(2):236−242 doi: 10.19303/j.issn.1008-0384.2021.02.015
引用本文: 吕新,刘兰英,陈丽华,等. 沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用 [J]. 福建农业学报,2021,36(2):236−242 doi: 10.19303/j.issn.1008-0384.2021.02.015
LYU X, LIU L Y, CHEN L H, et al. Visual LAMP for Salmonella Detection on Soil for Vegetable Farming [J]. Fujian Journal of Agricultural Sciences,2021,36(2):236−242 doi: 10.19303/j.issn.1008-0384.2021.02.015
Citation: LYU X, LIU L Y, CHEN L H, et al. Visual LAMP for Salmonella Detection on Soil for Vegetable Farming [J]. Fujian Journal of Agricultural Sciences,2021,36(2):236−242 doi: 10.19303/j.issn.1008-0384.2021.02.015

沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用

doi: 10.19303/j.issn.1008-0384.2021.02.015
基金项目: 福建省科技计划公益类专项(2018R1018-6、2019R1022-6);福建省农业科学院青年人才创新基金(YC2018-10)
详细信息
    作者简介:

    吕新(1980−),男,硕士,助理研究员,研究方向:农产品质量安全(E-mail:lux_ing@126.com

    通讯作者:

    李玥仁(1966−),男,博士,研究员,研究方向:农产品质量安全(E-mail:yuerenli@yeah.net

  • 中图分类号: S 19

Visual LAMP for Salmonella Detection on Soil for Vegetable Farming

  • 摘要:   目的  建立沙门氏菌可视化环介导恒温扩增体系(loop-mediated isothermal amplification, LAMP),并在快速检测蔬菜栽培土壤中应用,为蔬菜栽培土壤中沙门氏菌的防控提供技术支撑。  方法  根据沙门氏菌特有侵袭蛋白A (invA)基因序列设计可视化LAMP检测引物,并优化LAMP反应体系和反应条件,通过特异性试验、灵敏度试验和人工污染试验对LAMP检测方法进行验证,最后与国标检测方法对比检测蔬菜栽培土壤中沙门氏菌来验证该方法的准确性。  结果  优化后可视化LAMP检测方法,特异性试验结果显示除沙门氏菌的LAMP反应为阳性外,其余6个非沙门氏菌菌株的LAMP反应均为阴性,表明本LAMP检测方法具有良好的特异性;灵敏度试验结果显示本LAMP检测方法最低可以检测到7 CFU/25 μL的沙门氏菌DNA;人工污染试验结果显示本LAMP检测方法灵敏度达4×102 CFU·g−1,与国标检测方法对比检测蔬菜栽培土壤中沙门氏菌具有相同的准确性,但检测时间从5~7 d缩短至8 h内。  结论  本研究建立了快速、准确、灵敏的可视化恒温扩增体系,可以应用于蔬菜栽培土壤中沙门氏菌的快速检测。
  • 图  1  LAMP体系检测沙门氏菌

    注:A: LAMP产物琼脂糖电泳检测结果; B: LAMP产物钙黄绿素显色结果M: DL2 000 DNA Marker, 1: 鼠伤寒沙门氏菌, 2: 阴性对照

    Figure  1.  Detection of Salmonella using optimized LAMP assay

    Note: A: LAMP products shown on 2.0% agarose gel electrophoresis; B: LAMP products after calcein staining; M: DL2 000 DNA marker; 1: S. typhimurium; 2: Negative control.

    图  2  LAMP方法检测沙门氏菌的特异性

    注:A:LAMP产物琼脂糖电泳检测结果,B:LAMP产物钙黄绿素显色结果;M:DL2 000 DNA Marker,1:阴性对照,2:鼠伤寒沙门氏菌,3:金黄色葡萄球菌,4:大肠埃希氏菌,5:产气肠杆菌,6:大肠埃希氏菌O157:H7,7:单核细胞增生李斯特氏菌,8:英诺克李斯特氏菌

    Figure  2.  Specificity of LAMP assay in detecting Salmonella

    Note: A: LAMP products shown on 2% agarose gel electrophoresis; B: LAMP products after calcein staining; M: DL2 000 DNA marker; 1: Negative control; 2: S. typhimurium; 3: Staphylococcus aureus; 4: Escherichia coli; 5: Enterobacter aerogenes; 6: E. coli O157:H7; 7: Listeria monocytogenes; 8: Listeria innocua.

    图  3  LAMP方法检测沙门氏菌的灵敏度

    注:A:LAMP产物琼脂糖电泳检测结果,B:LAMP产物钙黄绿素显色结果;M:DL2 000 DNA Marker,1:阴性对照,2~8:每管反应体系中分别含有7×105 CFU、7×104 CFU、7×103 CFU、7×102 CFU、7×101 CFU、7 CFU、7 ×10−1 CFU

    Figure  3.  Sensitivity of LAMP assay in detecting Salmonella

    Note: A: LAMP products shown on 2% agarose gel electrophoresis; B: LAMP products after calcein staining; M: DL2 000 DNA marker; 1: Negative control; 2-8: Amplified products using DNA at concentration of 7×105 CFU, 7×104 CFU, 7×103 CFU, 7×102 CFU, 7×101 CFU, 7 CFU, and 0.7 CFU, respectively, in LAMP reaction tube.

    图  4  LAMP方法检测人工污染沙门氏菌的栽培土壤样品

    注:A:LAMP产物琼脂糖电泳检测结果;B:LAMP产物钙黄绿素显色结果;M:DL2 000 DNA Marker,1:阴性对照,2~8:不同含量人工污染沙门氏菌的栽培土壤样品扩增产物(0~1×105 CFU·g−1

    Figure  4.  LAMP assay for detecting artificially inoculated Salmonella in soil samples

    Note: a: LAMP products shown on 2% agarose gel electrophoresis; B: LAMP products after calcein staining; M: DL2 000 DNA marker; 1: Negative control; 2-8: Amplified products on different concentrations of Salmonella in artificially inoculated soil samples (0-1×105 CFU·g−1).

    表  1  供试菌株

    Table  1.   Bacteria used for testing

    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
    鼠伤寒沙门氏菌 Salmonella typhimurium 1.1174 CGMCC
    金黄色葡萄球菌 Staphylococcus aureus 25923 ATCC
    大肠埃希氏菌 Escherichia coli 25922 ATCC
    产气肠杆菌 Enterobacter aerogenes 1.0876 CGMCC
    大肠埃希氏菌O157:H7 Escherichia coli O157:H7 10907 CICC
    单核细胞增生李斯特氏菌 Listeria monocytogenes 1.9136 CGMCC
    英诺克李斯特氏菌 Listeria innocua 1.7730 CGMCC
    下载: 导出CSV

    表  2  环介导等温扩增引物

    Table  2.   Primers of LAMP

    引物
    Primer
    序列
    Sequence
    FIP 5′-GCGCGGCATCCGCATCAATATGCCCGGTAAACA
    GATGAGT-3′
    BIP 5′-GAACGGCGAAGCGTACTGGACATCGCACCGTC
    AAAGGAA-3′
    F3 5′-CGGCCCGATTTTCTCTGG-3′
    B3 5′-CGGCAATAGCGTCACCTT-3′
    Loop F 5′-GGCCTTCAAATCGGCATCAAT-3′
    Loop B 5′-AAGGGAAAGCCAGCTTTACG-3′
    下载: 导出CSV

    表  3  2种方法对比检测蔬菜栽培土壤沙门氏菌污染结果

    Table  3.   Salmonella detection in soil by two different methods

    方法
    Method
    土壤样品
    Soil samples
    阳性
    Positive
    阴性
    Negative
    国标方法 National standard method41239
    LAMP方法 LAMP method41239
    下载: 导出CSV
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  • 收稿日期:  2020-09-16
  • 修回日期:  2020-11-23
  • 网络出版日期:  2021-02-08
  • 刊出日期:  2021-02-28

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