基于叠氮溴化丙锭与恒温核酸扩增技术快速检测亚致死状态食源金黄色葡萄球菌

建立一种将荧光染料叠氮溴化丙锭（propidium monoazide，PMA）与环介导等温扩增（loop-mediatedisothermal amplification，LAMP）技术相结合的方法，用于快速高效检测金黄色葡萄球菌（Staphylococcusaureus）。同时，采用人工污染金黄色葡萄球菌的速冻水饺和奶粉作为食品样品，研究PMA-LAMP方法的检测灵敏度。结果表明，PMA溶液质量浓度3 μg/mL，650 W卤素灯下曝光5 min，PMA能够完全抑制1.2×107 copies/mL金黄色葡萄球菌死菌核酸扩增。PMA-LAMP方法能够在恒温65 ℃、60 min内完成对亚致死型金黄色葡萄球菌特异性nuc基因的特异性检测，其对亚致死状态金黄色葡萄球菌的检出限为34 CFU/mL，对食物样品速冻水饺和奶粉的检出限分别为17 CFU/mL和1.70×102 CFU/mL。建立的PMA-LAMP方法可以有效检测亚致死态金黄色葡萄球菌，提供了一种新的检测技术和解决方案。

Establishment of PMA-LAMP Method for Rapid Detection of Staphylococcus aureus with Sublethal Injury

In this study, a loop-mediated isothermal amplification (LAMP) assay combined with propidium monoazide (PMA) treatment was developed for the rapid and efficient detection of Staphylococcus aureus with sublethal injury. The sensitivity of PMA-LAMP was investigated by artificially contaminating food samples including frozen dumplings and milk powder with S. aureus. The results showed that a concentration of 3 μg/mL PMA was optimal for completely restraining the amplification of 1.2 × 107 copies/mL of DNA from dead S. aureus which was exposed to a 650 W halogen lamp for 5 min. PMA-LAMP could achieve the specific detection of the unique nuc gene of S. aureus with sublethal injury at a constant temperature of 65 ℃ within 60 min with a sensitivity of 34 CFU/mL. In addition, the PMA-LAMP assay was proved efficient by using it to detect the viable cells in artificially contaminated samples, with a sensitivity of 17 CFU/mL for artificially contaminated milk powder and 1.70 × 102 CFU/mL in artificially contaminated frozen dumplings. It can be concluded that the PMA-LAMP assay can efficiently detect S. aureus with sublethal injury and provide a new technique for detecting S. aureus in food samples.