沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用

吕新; 刘兰英; 陈丽华; 李玥仁

doi:10.19303/j.issn.1008-0384.2021.02.015

沙门氏菌LAMP检测方法的建立及其在蔬菜栽培土壤中的应用

Visual LAMP for Salmonella Detection on Soil for Vegetable Farming

摘要

摘要:
目的建立沙门氏菌可视化环介导恒温扩增体系（loop-mediated isothermal amplification, LAMP），并在快速检测蔬菜栽培土壤中应用，为蔬菜栽培土壤中沙门氏菌的防控提供技术支撑。
方法根据沙门氏菌特有侵袭蛋白A (invA)基因序列设计可视化LAMP检测引物，并优化LAMP反应体系和反应条件，通过特异性试验、灵敏度试验和人工污染试验对LAMP检测方法进行验证，最后与国标检测方法对比检测蔬菜栽培土壤中沙门氏菌来验证该方法的准确性。
结果优化后可视化LAMP检测方法，特异性试验结果显示除沙门氏菌的LAMP反应为阳性外，其余6个非沙门氏菌菌株的LAMP反应均为阴性，表明本LAMP检测方法具有良好的特异性；灵敏度试验结果显示本LAMP检测方法最低可以检测到7 CFU/25 μL的沙门氏菌DNA；人工污染试验结果显示本LAMP检测方法灵敏度达4×10² CFU·g⁻¹，与国标检测方法对比检测蔬菜栽培土壤中沙门氏菌具有相同的准确性，但检测时间从5～7 d缩短至8 h内。
结论本研究建立了快速、准确、灵敏的可视化恒温扩增体系，可以应用于蔬菜栽培土壤中沙门氏菌的快速检测。

Abstract:
Objective A visual loop-mediated isothermal amplification (LAMP) technique for rapid detection of Salmonella in soil was developed to facilitate the prevention and control of the pathogens in vegetable fields.
Method Based on the sequence of the invasion protein A (invA) of Salmonella, LAMP primers were designed and reaction system optimized. Specificity and sensitivity of the methodology were challenged by artificial inoculation and verified by the National Standard Detection Method for Salmonella Detection in Vegetable Cultivation Soil.
Result The LAMP reacted positively on Salmonella but negatively on 6 non-Salmonella strains. It showed a minimum detection limit of 7 CFU·25 μL⁻¹ and a sensitivity of 4×10²CFU·g⁻¹ on the artificially inoculated Salmonella. Compared with the standard method, the LAMP exhibited same testing accuracy.
Conclusion The newly developed visual LAMP assay was rapid, accurate and sensitive for the detection of Salmonella in soil for vegetable farming.

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