环介导恒温扩增结合纳米生物传感器快速检测类鼻疽伯克霍尔德菌的方法及临床应用

王毅 王晓霞 王立程 韩丽梅 李沙 符晓莹 李欢 陈海 朱雄

王毅, 王晓霞, 王立程, 韩丽梅, 李沙, 符晓莹, 李欢, 陈海, 朱雄. 环介导恒温扩增结合纳米生物传感器快速检测类鼻疽伯克霍尔德菌的方法及临床应用[J]. 疾病监测, 2021, 36(8): 831-836. doi: 10.3784/jbjc.202108010422
引用本文: 王毅, 王晓霞, 王立程, 韩丽梅, 李沙, 符晓莹, 李欢, 陈海, 朱雄. 环介导恒温扩增结合纳米生物传感器快速检测类鼻疽伯克霍尔德菌的方法及临床应用[J]. 疾病监测, 2021, 36(8): 831-836. doi: 10.3784/jbjc.202108010422
Wang Yi, Wang Xiaoxia, Wang Licheng, Han Limei, Li Sha, Fu Xiaoying, Li Huan, Chen Hai, Zhu Xiong. Development and clinical application of loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor for rapid detection of Burkholderia pseudomallei[J]. Disease Surveillance, 2021, 36(8): 831-836. doi: 10.3784/jbjc.202108010422
Citation: Wang Yi, Wang Xiaoxia, Wang Licheng, Han Limei, Li Sha, Fu Xiaoying, Li Huan, Chen Hai, Zhu Xiong. Development and clinical application of loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor for rapid detection of Burkholderia pseudomallei[J]. Disease Surveillance, 2021, 36(8): 831-836. doi: 10.3784/jbjc.202108010422

环介导恒温扩增结合纳米生物传感器快速检测类鼻疽伯克霍尔德菌的方法及临床应用

doi: 10.3784/jbjc.202108010422
基金项目: 海南省重点研发计划(No. ZDYF2019149,No. ZDYF2017163);三亚市医疗卫生科技创新项目(No. 2019YW20)
详细信息
    作者简介:

    王毅,男,贵州省遵义市人,助理研究员,主要从事病原诊断及感染组学研究

    通讯作者:

    王毅,Tel: 010–85695548,Email: wildwolf0101@163.com

    朱雄,Tel: 0898–88021115,Email:zhuxiong6@163.com

  • 中图分类号: R211; R378; R446.5

Development and clinical application of loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor for rapid detection of Burkholderia pseudomallei

Funds: This work was supported by the fund for the Key Research and Development Program of Hainan Province (No. ZDYF2019149, No.Z DYF2017163) and the Science and Technology Innovation Project of Medical Health in Sanya (No. 2019YW20)
More Information
  • 摘要:   目的   建立一种环介导恒温扩增(LAMP)结合纳米生物传感器(LFB)快速检测类鼻疽伯克霍尔德菌的快速方法,为临床检测提供依据。  方法   针对类鼻疽伯克霍尔德菌的Ⅲ型分泌系统基因设计LAMP特异性引物,建立LAMP方法,通过纳米生物传感判断结果,并应用临床样本对该方法进行评价。  结果   LAMP-LFB检测的最佳扩增条件为67 ℃反应40 min,对类鼻疽伯克霍尔德菌纯培养物DNA灵敏度达到100 fg,对258株菌株进行检测,包括227株类鼻疽伯克霍尔德菌和31株非类鼻疽伯克霍尔德菌,特异性为100%。 应用于46个临床样本检测时,与传统分离培养方法相比,达到100%的诊断准确率。 LAMP-LFB整个检测过程可在1 h内完成,包括15 min DNA制备,40 min LAMP扩增,2 min的LFB结果判读。  结论   本研究建立的LAMP-LFB技术是一种快速、灵敏、特异的检测类鼻疽伯克霍尔德菌的方法,可作为类鼻疽基础、现场和临床实验室诊断的潜在工具。
  • 图  1  用于设计LAMP扩增引物的TSS1(型分泌系统)序列位置和方向示意

    注:右箭头代表原序列,左箭头代表反向互补序列

    Figure  1.  Sequence and location of TTS1(Type secretion systems)used to design multiple cross displacement amplification primers

    图  2  LAMP扩增结果和验证结果

    注:A. 特异性核酸扩增指示剂判读结果;B. LFB判读结果;A1与B1(类鼻疽伯克霍尔德菌模板BPC-066 DNA)阳性扩增;A2与B2(沙门菌模板DNA)阴性扩增;A3与B3(单增李斯特菌模板DNA)阴性扩增;A4与B4(无菌水)阴性扩增

    Figure  2.  Confirmation and verification of B. pseudomallei-LAMP products

    图  3  LAMP 引物的最佳反应温度测试

    注:阈值为0.1,浊度>0.1为阳性扩增。类鼻疽伯克霍尔德菌BPC-066 DNA浓度为10 pg/uL。在反应在恒温条件下(61 ~ 68 ℃,以1 ℃为间隔)得了8张动态曲线图(1 ~ 8)。如图从66 ℃ ~ 68 ℃显示出更快速率的扩增

    Figure  3.  Optimal temperature for B. pseudomallei-LAMP primer set

    图  4  LAMP-LFB检测类鼻疽伯克霍尔德菌的灵敏度

    注:1~8.类鼻疽伯克霍尔德菌BPC-066模板DNA浓度为10 ng、10 pg、1 pg、100 fg、10 fg、1 fg、100 ag/μL和空白对照。浓度10 ng ~ 100 fg观察到阳性扩增

    Figure  4.  Sensitivity of LAMP-LFB assay using serially diluted genomic templates with B. pseudomallei

    图  5  LAMP-LFB技术的最佳反应时间测试结果

    注:A. 20 min;B. 30 min;C. 40 min;D. 50 min扩增效果

    Figure  5.  Optimal duration of time required for LAMP-LFB assay

    图  6  LAMP-LFB法特异性检测评价

    注:1. 空白对照;2 ~ 21. 铜绿假单胞菌、肺炎克雷伯菌、脑膜炎奈瑟菌、鲍曼不动杆菌、金黄色葡萄球菌、表皮葡萄球菌、腐生葡萄球菌、蜡样芽孢杆菌、热带念珠菌、白色念珠菌、粪肠球菌、屎肠球菌、产肠毒素大肠埃希菌、沙门菌、志贺菌、单增李斯特菌、肺炎链球菌、猪葡萄球菌、副溶血弧菌、嗜麦芽寡养单胞菌;22 ~ 24. 泰国伯克霍尔德菌;25 ~ 26. 洋葱伯克霍尔德菌;27 ~ 28. 类鼻疽伯克霍尔德菌

    Figure  6.  Specificity of LAMP-LFB assay using different bacterial strains

    表  1  引物序列及修饰信息

    Table  1.   Primers used in this study

    引物序列及修饰(5'~3')长度基因
    F3GAGATCGCCTGCCTGG16 ntⅢ型分泌系统(TTS1
    B3TGCCGGTGTCGGTTCC16 nt
    FIPTGTTCTCGGCATGCCCGCCGACAGACGGCGCTTCA 35 mer
    FIPaFITC-TGTTCTCGGCATGCCCGCCGACAGACGGCGCTTCA 35 mer
    BIPCCATCCGCCTCGATTGCGCATCTGTTGCTAGCGGATTGT 39 mer
    LFCAACCACAGCAACGGAA17 nt
    LFbBiotin-CAACCACAGCAACGGAA17 nt
    LBGCAACGCACTGCCTG15 nt
      注:a. 该引物用于LAMP-LFB检测体系时,在5'端标记荧光素;b. 该引物用于LAMP-LFB检测体系时,在5'端标记生物素;mer. 单体单元;nt. 核苷酸
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  • 收稿日期:  2021-08-01
  • 网络出版日期:  2021-08-23
  • 刊出日期:  2021-08-31

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