黄疸出血群钩端螺旋体LAMP-LFB快速检测方法的建立与应用

王毅 杨幸贵 刘英 黄俊飞 王铭 营夏 谭勤琴 胡勇 李世军

王毅, 杨幸贵, 刘英, 黄俊飞, 王铭, 营夏, 谭勤琴, 胡勇, 李世军. 黄疸出血群钩端螺旋体LAMP-LFB快速检测方法的建立与应用[J]. 疾病监测, 2022, 37(5): 646-651. doi: 10.3784/jbjc.202108030429
引用本文: 王毅, 杨幸贵, 刘英, 黄俊飞, 王铭, 营夏, 谭勤琴, 胡勇, 李世军. 黄疸出血群钩端螺旋体LAMP-LFB快速检测方法的建立与应用[J]. 疾病监测, 2022, 37(5): 646-651. doi: 10.3784/jbjc.202108030429
Wang Yi, Yang Xinggui, Liu Ying, Huang Junfei, Wang Ming, Ying Xia, Tan Qinqin, Hu Yong, Li Shijun. Development and application of loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for rapid detection of Leptospira interrogans serogroup icterohaemorrhagiae[J]. Disease Surveillance, 2022, 37(5): 646-651. doi: 10.3784/jbjc.202108030429
Citation: Wang Yi, Yang Xinggui, Liu Ying, Huang Junfei, Wang Ming, Ying Xia, Tan Qinqin, Hu Yong, Li Shijun. Development and application of loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for rapid detection of Leptospira interrogans serogroup icterohaemorrhagiae[J]. Disease Surveillance, 2022, 37(5): 646-651. doi: 10.3784/jbjc.202108030429

黄疸出血群钩端螺旋体LAMP-LFB快速检测方法的建立与应用

doi: 10.3784/jbjc.202108030429
基金项目: 国家自然科学基金(No. 81760366, No. 81960622);贵州省高层次创新型人才培养项目[No. 黔科合(2016)4021]
详细信息
    作者简介:

    王毅,男,贵州省遵义市人,助理研究员,主要从事病原快速鉴别技术研究及感染组学研究,Email:wildwolf0101@163.com

    通讯作者:

    李世军,Tel:0851–86827001,Email:zjumedjun@163.com

  • 中图分类号: R211; R377+.5

Development and application of loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for rapid detection of Leptospira interrogans serogroup icterohaemorrhagiae

Funds: This study was supported by the National Natural Science Foundation of China (No. 81760366, No. 81960622) and fund for Guizhou Province High-level Innovative Talent Training Project [No. Qiankehe (2016) 4021]
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  • 摘要:   目的   基于环介导恒温扩增(LAMP)结合纳米生物传感条(LFB)技术,建立并评价黄疸出血群钩端螺旋体(钩体)的LAMP-LFB快速检测方法。  方法   以黄疸出血群钩体O抗原基因簇中的糖基转移酶基因(gtf)为检测靶标并设计特异性LAMP引物。 通过对LAMP引物的特定标记(FIP-FAM和LF-Biotin)和优化试验,建立LAMP-LFB快速检测方法,并分析其灵敏度和特异性。 应用建立的LAMP-LFB、普通PCR方法和显微凝集试验(MAT),分别对53株钩体分离菌株进行鉴定,比较分析鉴定结果并评价LAMP-LFB方法的实用性。   结果   灵敏度和特异性试验显示,LAMP-LFB方法可检出黄疸出血群赖型56601基因组DNA的最低浓度为100 fg/µL,检测特异性为100%,与其余血清群和非钩体菌株核酸无交叉反应。 菌株鉴定结果显示,LAMP-LFB与MAT鉴定结果完全一致,符合率为100%,其敏感性高于PCR方法。 此外,LAMP扩增产物经LFB验证,可直接通过观察检测线(TL)和质控线(CL)进行结果判定。  结论   基于gtf基因建立的LAMP-LFB检测技术方便、快捷且重复性好,可快速、灵敏、特异地鉴定黄疸出血群钩体菌株,能够作为有价值的黄疸出血群钩体菌株快速鉴别或诊断方法。
  • 图  1  环介导恒温扩增结合纳米生物传感条技术检测黄疸出血群钩端螺旋体的灵敏度

    注:A. LFB生物传感条验证;B. MG 可视化反应;C. 1.5% 琼脂糖凝胶电泳;D. 实时浊度仪监测;A/B/C/D中1~8. 10 ng/µL、1 ng/µL、100 pg/µL、10 pg/µL、1 pg/µL、100 fg/µL、10 fg/µL、1 fg/µL;9. 空白对照(水);M. 100 bp ladder

    Figure  1.  Sensitivity of LAMP-LFB assay for detection of Leptospira interrogans serogroup icterohaemorrhagiae

    图  2  环介导恒温扩增结合纳米生物传感条技术检测黄疸出血群钩端螺旋体的特异性

    注:1. 黄疸出血群赖型56601;2~5. 黄疸出血群分离菌株;6. 爪哇群56602;7. 犬群56603;8. 拜伦群56604;9. 致热群56605;10. 秋季群56606;11. 澳洲群56607;12. 波摩那群56608;13. 流感伤寒群56609;14. 七日热群56610;15. 赛罗群56612;16. 明尼群56613;17. 巴达维亚群56615;18. 塔拉索夫群56635;19. 曼耗群56655;20. 双曲钩体57601;21~26. 结核分枝杆菌(H37Rv)、牛分枝杆菌、沙门菌、肺炎克雷伯菌、单核细胞增生李斯特菌、大肠埃希菌;27. 阴性对照

    Figure  2.  Specificity of LAMP-LFB assay for detection of Leptospira interrogans serogroup icterohaemorrhagiae

    表  1  用于扩增gtf基因的引物

    Table  1.   Primers used for amplification of gene gtf

    用途名称序列(5′~3′)长度(bp)
    LAMPF3GAAAACAAAATATTCTATTGGGC23
    B3CTCAGAGGAATTTAAATGGTT21
    FIP*FAM-CCTCGAGTACATTCCATTGATGTTT-AAAAAACGACCGATAGAATTGC47
    BIPGAGTGATTTAAATTGTGCAGGGATT-GAAAAGTTTATTTACGGAAGCT47
    LF*Biotin-ATATGCACCAATAAATGAAACA22
    LBGCTTGGTTGGTTCTCATAA19
    PCRFCAGGATTTATCGGTGGTACT20
    RTTACGAAAAGAAGTTCCTTGAA22
      注:FAM为6-羧基荧光素
    下载: 导出CSV

    表  2  53株钩端螺旋体分离株的鉴定结果

    Table  2.   Identification results of 53 isolates of Leptospira

    检测方法阳性阴性
    PCR494
    LAMP-LFB530
    MAT530
    下载: 导出CSV
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  • 收稿日期:  2021-08-03
  • 网络出版日期:  2022-01-14
  • 刊出日期:  2022-05-31

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