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

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

王毅^1,,
杨幸贵^{2, 3},
刘英³,
黄俊飞³,
王铭³,
营夏^{2, 3},
谭勤琴^{2, 3},
胡勇²,
李世军^{2, 3, ,}

1.
首都儿科研究所实验中心, 北京 100020
2.
贵州医科大学公共卫生学院, 环境污染与疾病监控教育部重点实验室, 贵州贵阳 550025
3.
贵州省疾病预防控制中心, 贵州贵阳 550004

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
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出版历程
- 收稿日期: 2021-08-03
- 网络出版日期: 2022-01-14
- 刊出日期: 2022-05-31

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

Wang Yi^1
,,
Yang Xinggui^{2, 3},
Liu Ying³,
Huang Junfei³,
Wang Ming³,
Ying Xia^{2, 3},
Tan Qinqin^{2, 3},
Hu Yong²,
Li Shijun^{2, 3
, ,}

1.
Experimental Research Center, Capital Institute of Pediatrics, Beijing 100020, China
2.
School of Public Health, the Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang 550025, Guizhou, China
3.
Guizhou Center for Disease Control and Prevention, Guiyang 550004, Guizhou, China

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]

More Information

Corresponding author: Li Shijun, Email: zjumedjun@163.com

摘要

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。
- 环介导恒温扩增 /
- 纳米生物传感 /
- 钩端螺旋体 /
- 黄疸出血群
Abstract: Objective To develop and evaluate a rapid detection assay for Leptospira interrogans serogroup icterohaemorrhagiae based on loop-mediated isothermal amplification (LAMP) combined with nanoparticle-based lateral flow biosensor (LFB). Methods The LAMP primers targeting the glycotransferase gene (gtf) in the O-antigen gene cluster of L.serogoup icterohaemorrhagiae were designed. After specific label of primers (FIP-FAM and LF-Biotin) and condition optimization, we evaluated the sensitivity, specificity and feasibility of gtf-LAMP-LFB assay. A total of 53 strains of Leptospira were identified by using LAMP-LFB assay, PCR and microscopic agglutination test (MAT), and the results were analyzed to evaluate the practicality of LAMP-LFB assay. Results Our data showed that the detection limit of the assay was 100 fg/μL for genomic DNA of reference strain of L. icterohaemorrhagiae (56601), and the specificity was 100% because there were no cross reactions with nucleic acids of other Leptospira serogroups and non-Leptospira strains. For the application examination, LAMP-LFB and MAT identification results were completely consistent, while the sensitivity of LAMP-LFB assay was higher than PCR. In addition, the color of CL and TL bands could be observed directly to determine the results by using LFB to detect LAMP amplicons. Conclusion The LAMP-LFB assay developed based on LAMP technique has high repeatability, sensitivity and specificity, which can be used for the rapid and accurate identification of the strains of L.interrogans serogroup icterohaemorrhagiae, and can be used as a potential screening and diagnosis tool for L.interrogans serogroup icterohaemorrhagiae.
- Loop-mediated isothermal amplification /
- Lateral flow biosensor /
- Leptospira /
- Serogoup icterohaemorrhagiae

HTML全文

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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）
LAMP	F3	GAAAACAAAATATTCTATTGGGC	23
	B3	CTCAGAGGAATTTAAATGGTT	21
	FIP*	FAM-CCTCGAGTACATTCCATTGATGTTT-AAAAAACGACCGATAGAATTGC	47
	BIP	GAGTGATTTAAATTGTGCAGGGATT-GAAAAGTTTATTTACGGAAGCT	47
	LF*	Biotin-ATATGCACCAATAAATGAAACA	22
	LB	GCTTGGTTGGTTCTCATAA	19
PCR	F	CAGGATTTATCGGTGGTACT	20
	R	TTACGAAAAGAAGTTCCTTGAA	22
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
PCR	49	4
LAMP-LFB	53	0
MAT	53	0

下载: 导出CSV

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