检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用

doi:10.11843/j.issn.0366-6964.2022.06.017

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (6): 1841-1848.doi: 10.11843/j.issn.0366-6964.2022.06.017

检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用

江玲丽¹, 高有领^2*, 胡兴娟³, 章先⁴, 杨云凯⁴, 魏华¹, 方维焕⁴

1. 宁波卫生职业技术学院，宁波 315100;
2. 浙江万里学院，宁波 315100;
3. 舟山海关综合技术服务中心，舟山 316021;
4. 浙江大学动物预防医学研究所，杭州 310058

收稿日期:2021-08-31 出版日期:2022-06-23 发布日期:2022-06-25
通讯作者: 高有领，主要从事食品营养与安全研究，E-mail：gaoyl@zwu.edu.cn
作者简介:江玲丽(1980-)，女，浙江温岭人，博士，研究员，主要从事食品营养与安全研究，E-mail：jllgrace@163.com
基金资助:
浙江省自然科学基金项目(LY19C180001)；浙江省科技厅公益技术研究农业项目(2014C32047)

Development and Primary Application of Colloidal Gold-based Test Strip for Rapid Detection of Listeria monocytogenes LLO

JIANG Lingli¹, GAO Youling^2*, HU Xingjuan³, ZHANG Xian⁴, YANG Yunkai⁴, WEI Hua¹, FANG Weihuan⁴

1. Ningbo College of Health Sciences, Ningbo 315100, China;
2. Zhejiang Wanli University, Ningbo 315100, China;
3. Zhoushan Customs Integrated Technical Service Centre, Zhoushan 316021, China;
4. Zhejiang University Institute of Preventive Veterinary Medicine, Hangzhou 310058, China

Received:2021-08-31 Online:2022-06-23 Published:2022-06-25

摘要/Abstract

摘要： 产单核细胞李氏杆菌为重要的食源性病原菌，本研究旨在建立快速、灵敏、特异的检测方法，提高检测效率和检测通量。李氏杆菌溶血素(LLO)蛋白经原核表达后, 制备单克隆抗体, 测定单克隆抗体的亚型、效价及亲和力。柠檬酸钠法研制LLO胶体金试纸条，并对试纸条的特异性、敏感性、重复性、稳定性及模拟样品进行检测，并初步应用于实际海产品的检测。结果发现，制备的LLO单克隆抗体特异性强、稳定性高、重复性佳、灵敏度较高，亲和力好，亲和力常数为4.25×10⁸ L·mol^－1。人工污染样品试验表明灵敏度与纯细菌培养物基本一致，为3.0×10⁵CFU·mL^-1。将LLO胶体金试纸条应用于500份海产品的实际检测中，产单核细胞李氏杆菌的阳性检出率为2.20% (11/500)，略低于国标检测方法(GB4789.30—2010)的2.40%(12/500)和PCR检测方法的2.40%(12/500)，三者之间符合率为91.67%。另外缩短检测时间至15 min，检测效率大幅度提高。本研究建立的LLO胶体金检测方法可用于产单核细胞李氏杆菌的快速检测。

关键词: 产单核细胞李氏杆菌, 李氏杆菌溶血素, 单克隆抗体, 胶体金试纸, 海产品初步应用

Abstract: Listeria monocytogenes is an important food-borne pathogen. So it's important and urgent to establish a sensitive and specific method for its rapid detection. The monoclonal antibody of Listeriolysin O (LLO) was screened after prokaryotic expression of recombinant soluble protein His-LLO as antigen, followed by detection of the subtypes, titers and affinity of the antibody. Colloidal gold-based test strip for Listeria monocytogenes LLO was developed via sodium citrate method. Subsequently, the specificity, sensibility, repeatability and stability as well as the primary application of the strip in simulated samples and seafood samples were performed. The results showed that the monoclonal antibody of LLO was specific, stable, repeatable, relatively sensitive and had high affinity, with its affinity constant reaching to 4.25×10⁸ L·mol^-1. The sensibility of the LLO strip in the simulated samples was 3.0×10⁵CFU·mL^-1, which was consistant with that in the pure bacterial cultures. The positive detection of L.monocytogenes from 500 seafood samples was 2.20% (11/500) via LLO colloidal gold-based test strip, which was a bit lower than using standard method (2.40%, 12/500) and PCR detection method (2.40%, 12/500). The coincidence rate of L. monocytogenes positive detection via the aforementioned three methods was 91.67%. Besides, the detection time was deceased to 15 minutes, thereby the efficiency was increased to a greater extent. Therefore, the developed LLO colloidal gold-based test strip could be further used for the rapid detection of L. monocytogenes.

Key words: Listeria monocytogenes, Listeriolysin O, monoclonal antibody, colloidal gold-based test strip, primary application in seafood samples

中图分类号:

S855.1

江玲丽, 高有领, 胡兴娟, 章先, 杨云凯, 魏华, 方维焕. 检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用[J]. 畜牧兽医学报, 2022, 53(6): 1841-1848.

JIANG Lingli, GAO Youling, HU Xingjuan, ZHANG Xian, YANG Yunkai, WEI Hua, FANG Weihuan. Development and Primary Application of Colloidal Gold-based Test Strip for Rapid Detection of Listeria monocytogenes LLO[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1841-1848.

参考文献 20

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检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用

Development and Primary Application of Colloidal Gold-based Test Strip for Rapid Detection of Listeria monocytogenes LLO

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