重组酶介导等温核酸扩增方法快速检测土壤沙门氏菌

Rapid detection of Salmonella in soil by recombinase aided amplification method

摘要:

【背景】沙门氏菌(Salmonella)是一种可以引起人畜患病的致病菌，也是最主要的食源性细菌之一。土壤中的沙门氏菌可通过蔬菜等植物进入人体，引发食物中毒。但由于土壤性质及其他微生物的干扰，如何快速甄别土壤是否受到沙门氏菌的污染仍是一个难题。【目的】建立一种快速、灵敏检测土壤沙门氏菌的实时重组酶介导等温核酸扩增(Real-Time Recombinase Aided Amplification，RT-RAA)方法。【方法】针对沙门氏菌invA基因序列设计特异性引物和探针，构建含有invA基因待检片段的重组质粒，评价RT-RAA方法的灵敏度；分别以肠炎沙门氏菌、大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌的基因组DNA为模板，评价RT-RAA方法的特异性；RT-RAA方法用于番茄、生姜土壤中沙门氏菌的检测，同时用平板培养法进行验证。【结果】RT-RAA方法可用于重组质粒中invA基因片段的检测，在39 °C条件下，20 min内即可获得检测结果，最低检测质粒拷贝数为10拷贝/反应，而且与大肠杆菌、福氏志贺氏菌和金黄色葡萄球菌无交叉反应。土壤样品DNA的RT-RAA检测结果显示，供试番茄土已被沙门氏菌污染，而生姜土则没有，与平板培养结果一致。【结论】RT-RAA方法具有灵敏度高和特异性强的特点，可用于土壤沙门氏菌污染的快速检测。

Abstract:

[Background] Salmonella is a pathogenic bacterium that can cause human and animal diseases, and is also one of the most important foodborne bacteria. Salmonella in soil causes food poisoning in humans through plants such as vegetables. However, due to the nature of the soil and interference from other microorganisms, how to quickly identify whether the soil is contaminated with salmonella remains a challenge. [Objective] To develop a real-time recombinase aided amplification (RT-RAA) method for rapid and sensitive detection of Salmonella in soil. [Methods] A pair of specific primers and a probe were designed targeting the sequence of invA gene in Salmonella, and a recombinant plasmid containing invA gene fragment was constructed to evaluate the sensitivity of RT-RAA method. The specificity of RT-RAA method was performed by amplification of template DNA from standard strains including Salmonella enteritidis, Escherichia coli, Shigella flexneri and Staphylococcus aureus. RT-RAA method was used to detect Salmonella in soil of tomato and ginger, and plate culture method were used to verify the results. [Results] RT-RAA method can be used to detect invA gene fragment in the recombinant plasmid at a minimum copy number of 10 per reaction within 20 min at 39 °C, without cross-reaction with Escherichia coli, Shigella flexneri and Staphylococcus aureus. RT-RAA detection of the DNA in the tested soil samples showed that the tomato soil was contaminated with Salmonella, while the ginger soil was not, which was consistent with the results of plate culture. [Conclusion] RT-RAA method has the characteristics of high sensitivity and specificity, and can be used for rapid detection of soil salmonella contamination.