重组酶介导扩增技术快速检测食品中单核细胞增生李斯特菌

目的建立重组酶介导扩增技术(recombinase aided amplification,RAA)快速检测单核细胞增生李斯特菌的方法。方法以单核细胞增生李斯特菌的hlyA基因作为靶序列,设计引物和探针,通过构建含目的基因片段的重组质粒,评价RAA方法的灵敏度,通过检测沙门氏菌核酸、金黄色葡萄球菌核酸、副溶血性弧菌核酸、溶血性链球菌核酸、志贺氏菌核酸基因组DNA,评价其特异性。结果建立的RAA方法可在39℃、20 min内,检测重组质粒中hlyA基因片段,最低可检出的质粒拷贝数为10拷贝/反应,且与沙门氏菌、金黄色葡萄球菌、副溶血性弧菌、溶血性链球菌、志贺氏菌基因组DNA无交叉反应。结论建立的RAA方法具有灵敏度高和特异性强的特点,可用于单核细胞增生李斯特菌的快速检测。

Rapid detection of Listeria monocytogenes by recombinase aided amplification method

Objective To establish a recombinase aided amplification(RAA)method for detection of Listeria monocytogenes.Methods The primers and probe were designed according the specific hlyA gene sequence of Listeria monocytogenes.The sensitivity of RAA method was evaluated using a recombinant plasmid containing hlyA gene fragment.The specificity was performed by amplification of genomic DNA of Salmonella,Staphylococcus aureus,Vibrio Parahemolyticus,Streptococcus hemolyticus and Shigella.Results RAA method can be used to detect hlyA gene fragment in the recombinant plasmid at a copy number as low as 10 copies per reaction within 20 min at 39℃with no cross-reaction with Salmonella,Staphylococcus aureus,Vibrio Parahemolyticus,Streptococcus hemolyticus and Shigella.Conclusion RAA method has the characteristics of high sensitivity and specificity,and can be used for rapid detection of Listeria monocytogenes.