副结核分枝杆菌重组酶介导扩增-侧向流试纸条检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2024.07.043

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (7): 3255-3260.doi: 10.11843/j.issn.0366-6964.2024.07.043

副结核分枝杆菌重组酶介导扩增-侧向流试纸条检测方法的建立与初步应用

南岳(), 龙美贞(), 王元智, 刘一朵, 周向梅*()

中国农业大学动物医学院兽医公共卫生安全全国重点实验室, 北京 100193

收稿日期:2023-10-07 出版日期:2024-07-23 发布日期:2024-07-24
通讯作者: 周向梅 E-mail:2965008308@qq.com;1727293215@qq.com;zhouxm@cau.edu.cn
作者简介:南岳(2000-)，男，河南周口人，硕士，主要从事牛结核病防治研究，E-mail: 2965008308@qq.com
龙美贞(1998-)，女，土家族，重庆酉阳人，硕士，主要从事牛结核病防治研究，E-mail: 1727293215@qq.com
第一联系人：
南岳和龙美贞为同等贡献作者
基金资助:
“十四五”重点研发计划(2022YFD1800703)

Establishment and Preliminary Application of Recombinase-mediated Amplification-sidestream Detection for Detection of Mycobacterium paratuberculosis

Yue NAN(), Meizhen LONG(), Yuanzhi WANG, Yiduo LIU, Xiangmei ZHOU*()

National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Received:2023-10-07 Online:2024-07-23 Published:2024-07-24
Contact: Xiangmei ZHOU E-mail:2965008308@qq.com;1727293215@qq.com;zhouxm@cau.edu.cn

摘要/Abstract

摘要：

旨在建立一种副结核分枝杆菌(Mycobacterium avium subsp. paratuberculosis，MAP)精准快速的检测方法，结合重组酶介导扩增(recombinase-aid amplification, RAA)和侧向流试纸条(lateral flow dipstick, LFD)技术，以MAP的特异性多拷贝插入元件IS900设计引物和探针，通过优化反应时间和温度等条件，建立可用于MAP现场可视化检测的RAA-LFD方法。该检测方法在35 ℃条件下恒温反应30 min即可实现对MAP目的基因的有效扩增。试验结果显示：该方法可特异性地检测出MAP，不与其他常见的病原发生交叉反应；对MAP标准品的最低检测限可达到10 fg·μL^-1；使用该方法对149份牛奶样品进行检测，与传统的PCR相比，其敏感性为100%。综上，本研究成功建立了一种针对于MAP的精准快速的试纸条检测方法，并具有广泛的应用前景。

关键词: 副结核分枝杆菌, 重组酶介导扩增, 侧向流试纸条

Abstract:

To establish a rapid and accurate method for detection of Mycobacterium avium subsp. paratuberculosis (MAP), primers and probes were designed according to the specific multicopy insert element IS900 of MAP. A recombinase-aid amplification combined with lateral flow dipstick (RAA-LFD) method was established for visual detection of MAP in the field within 30 min at 35 ℃ after being optimized. The assay was specific with the target sequence of MAP with a detection limit up to 10 fg·μL^-1. One hundred and forty-nine stool samples were tested by this method, showing 100% sensitivity with the PCR. In short, the RAA-LFD assay developed in this study was a rapid, reliable method which can be broadly used.

Key words: Mycobacterium avium subsp. paratuberculosis, recombinase-aid amplification, lateral flow dipstick

中图分类号:

S852.618

南岳, 龙美贞, 王元智, 刘一朵, 周向梅. 副结核分枝杆菌重组酶介导扩增-侧向流试纸条检测方法的建立与初步应用[J]. 畜牧兽医学报, 2024, 55(7): 3255-3260.

Yue NAN, Meizhen LONG, Yuanzhi WANG, Yiduo LIU, Xiangmei ZHOU. Establishment and Preliminary Application of Recombinase-mediated Amplification-sidestream Detection for Detection of Mycobacterium paratuberculosis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3255-3260.

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参考文献 17

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引物/探针Primers/Probe	序列(5′ → 3′) Sequences
F	CGCTGCTGGAGTTGATTGCGGCGGTGAC
R	GCGTCTTTGGCGTCGGTCTTGCCTTCGC-Boitin
nfo-Probe	FAM-CGCTGCTGGAGTTGATTGCGGCGG-dSpacer- TGACGACGTTGGCCGATGGAGGCGAGGTCACGTGGGCGATCGACCTCAACGCCGGCGGC GCCGCGTTGCTGATCGCCTTGCTCATCGCTGCCGGGCAGCGGCTGCTTTATATTCCCGG GCGCACGGTCCATCACGCCGCGGGTAGTTACCGCGGCGAAGGCAAGACCGACGCCAAA GACGC-C3-spacer

副结核分枝杆菌重组酶介导扩增-侧向流试纸条检测方法的建立与初步应用

Establishment and Preliminary Application of Recombinase-mediated Amplification-sidestream Detection for Detection of Mycobacterium paratuberculosis

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RAA-LFD	PCR		总样本数 Total number of samples
RAA-LFD	阳性样本数 Number of positive samples	阴性样本数 Number of negative samples	总样本数 Total number of samples
阳性样本数 Number of positive samples	33	22	55
阴性样本数 Number of negative samples	0	94	94
总样本数 Total number of samples	33	116	149