可视化RPA-LFD技术快速检测猪链球菌

doi:10.11843/j.issn.0366-6964.2022.02.020

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (2): 538-547.doi: 10.11843/j.issn.0366-6964.2022.02.020

可视化RPA-LFD技术快速检测猪链球菌

张闪闪¹, 何斌¹, 李书光², 刘明成¹, 姜金庆¹, 胡建和¹, 雷连成³, 沈志强^2*, 夏小静^1*

1. 河南科技学院动物科技学院, 新乡 453003;
2. 山东省滨州畜牧兽医研究院, 滨州 256600;
3. 吉林大学动物医学学院, 长春 130062

收稿日期:2021-06-10 出版日期:2022-02-23 发布日期:2022-03-02
通讯作者: 夏小静,主要从事动物病原致病机理与快速检测技术及新兽药(抗菌肽)研发,E-mail:quik500@163.com;沈志强,主要从事预防兽医学研究,E-mail:bzshenzq@163.com
作者简介:张闪闪(1997-),女,河南开封人,硕士生,主要从事分子病原学研究,E-mail:1812099876@qq.com;何斌(1996-),河南南阳人,硕士生,主要从事分子病原学研究,E-mail:Hebin@stu.hist.edu.cn;李书光(1982-),山东惠民人,副研究员,博士,主要从事分子病原学研究,E-mail:lshug@163.com
基金资助:
国家自然科学基金（32172876；31972715；32072823）；河南省重点研发与推广专项（科技攻关）-农业领域项目（202102110101）；河南省青年人才托举工程项目（2021HYTP038）；河南省高等学校青年骨干教师培养计划项目（2020 GGJS163）；河南省高校科技创新团队支持计划（20IRTSTHN025）

Rapid Detection of Streptococcus suis with Visual RPA-LFD Technology

ZHANG Shanshan¹, HE Bin¹, LI Shuguang², LIU Mingcheng¹, JIANG Jinqing¹, HU Jianhe¹, LEI Liancheng³, SHEN Zhiqiang^2*, XIA Xiaojing^1*

1. College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China;
2. Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China;
3. College of Veterinary Medicine, Jilin University, Changchun 130062, China

Received:2021-06-10 Online:2022-02-23 Published:2022-03-02

摘要/Abstract

摘要： 旨在建立一种可视化、操作简便的猪链球菌快速检测技术。针对猪链球菌种特异性基因谷氨酸脱氢酶（gdh）序列设计特异性引物，建立可同时检测出所有血清型的重组酶聚合酶扩增结合侧流层析试纸条（RPA-LFD）检测技术，优化反应温度与反应时间，评价其特异性与灵敏度，同时利用该法对45例疑似猪链球菌感染标本进行检测。结果显示：猪链球菌RPA-LFD法检测灵敏度可达100拷贝·μL^-1，优于常规PCR方法，且不与其他常见病原菌发生交叉反应。扩增反应可在较宽温度范围（30~45℃）内高效进行，20~30 min可完成检测。利用临床45例疑似猪链球菌感染样本对检测体系进行评估，其检出率高于细菌分离与常规PCR方法，具有较强的实用性。本研究建立的猪链球菌RPA-LFD检测方法具有强特异性、高灵敏度、易于操作的特点，同时不依赖于仪器、专业操作人员，适用于野外及现场检测。

关键词: 猪链球菌, 重组酶聚合酶等温扩增, 侧流层析试纸条, 快速检测

Abstract: Here, we describe the development of a visual and easy-to-operate rapid detection technology for Streptococcus suis. Specific primers were designed for the sequence of the species-specific gene glutamate dehydrogenase (gdh) of S. suis. The recombinant polymerase amplification combined with a lateral flow dipstick (RPA-LFD) detection technology was established, which can detect all serotypes of S. suis simultaneously. The reaction temperature and time were optimized, and its specificity and sensitivity were evaluated. At the same time, 45 samples of suspected S. suis infection were detected by this method. The results showed that the detection sensitivity of S. suis RPA-LFD method can reach 100 copies·μL^-1, which is superior to the conventional PCR method and does not cross-react with other common pathogens. The amplification reaction can be carried out efficiently in a wide temperature range (30-45℃), and the detection can be completed in 20-30 min. Using 45 clinical samples of suspected S. suis infection to evaluate the detection system, the detection rate of RPA-LFD is higher than that of bacterial isolation and conventional PCR method, indicating that it has strong practicability. The RPA-LFD detection method of S. suis established in this study has the characteristics of strong specificity, high sensitivity, easy operation. At the same time, it does not rely on instruments and professional operators, and is suitable for field and on-site detection.

Key words: Streptococcus suis, recombinase polymerase amplification, lateral flow dipstick, rapid detection

中图分类号:

S852.61

张闪闪, 何斌, 李书光, 刘明成, 姜金庆, 胡建和, 雷连成, 沈志强, 夏小静. 可视化RPA-LFD技术快速检测猪链球菌[J]. 畜牧兽医学报, 2022, 53(2): 538-547.

ZHANG Shanshan, HE Bin, LI Shuguang, LIU Mingcheng, JIANG Jinqing, HU Jianhe, LEI Liancheng, SHEN Zhiqiang, XIA Xiaojing. Rapid Detection of Streptococcus suis with Visual RPA-LFD Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 538-547.

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可视化RPA-LFD技术快速检测猪链球菌

Rapid Detection of Streptococcus suis with Visual RPA-LFD Technology

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