摘要:
目的:建立一种设备要求简单、结果可视化的用于定性检测畜禽肉中金黄色葡萄球菌活菌的快速检测方法。方法:通过对羟基萘酚蓝(hydroxynaphthol blue,HNB)浓度、叠氮溴化丙啶(propidium monoazide,PMA)浓度等的优化,结合环介导恒温扩增技术(loop-mediated isothermal amplification,LAMP),建立一种PMA-HNB-LAMP联用的可视化的用于快速检测畜禽肉中金黄色葡萄球菌活菌的方法,对其特异性、灵敏度进行测试,并用于人工污染样品和实际样品的检测。结果:本研究成功建立了LAMP扩增体系,并通过添加210 μmol/L HNB到LAMP扩增体系中,得到了差异明显的阴阳性结果。建立的HNB-LAMP法特异性强,其纯菌液灵敏度为104 CFU/mL,样品基质灵敏度为105 CFU/mL。本研究中用于处理样品的PMA浓度为10 μg/mL,处理条件为室温下避光孵育5 min,曝光15 min。在检测人工污染的样品时,PMA-HNB-LAMP法和传统微生物法检测结果一致。结论:本研究成功建立了可快速检测畜禽肉中金黄色葡萄球菌活菌的可视化PMA-HNB-LAMP法。
Abstract:
Objective:To establish a fast detection method with simple equipment requirements and visual results for Staphylococcus aureus in livestock and poultry meat. Methods:A visualized method of PMA-HNB-LAMP for rapid detection of Staphylococcus aureus in livestock and poultry meat was established by optimizing the concentration of HNB,PMA. In this study,the specificity,sensitivity,simulated pollution experiment and practical application effect were tested. Results:A LAMP amplification system was successfully established. By adding 210 μmol/L HNB to the LAMP amplification system,significant negative and positive results could be obtained. The HNB-LAMP method established in this study had strong specificity,the sensitivity of the pure bacterial solution of this method was 104 CFU/mL,and the sensitivity of the sample matrix was 105 CFU/mL.Through the optimization of PMA concentration and processing conditions,it was determined to use PMA of 10 μg/mL to treat samples by incubating in darkness for 5 min and exposing for 15 min at the room temperature. The results of PMA-HNB-LAMP and traditional microbiological methods were consistent in the detection of artificially polluted samples. Conclusions:A PMA-HNB-LAMP method for the detection of living Staphylococcus aureus in livestock and poultry meat was successfully established in this research.
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