College of Chemical Engineering, Shijiazhuang University, Shijiazhuang, Hebei 050035, People's Republic of China.
College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang, Hebei 050018, People's Republic of China.
J Food Prot. 2022 Mar 1;85(3):414-423. doi: 10.4315/JFP-21-272.
Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and Pseudomonas lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP-amplified products were approximately 90.0 and 88.0°C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU per reaction in pure culture and 2.2 CFU per reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Real-time LAMP was used to test 200 raw milk samples for the presence of TAP-harboring P. fluorescens in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5 to 7 days, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.
荧光假单胞菌产生的嗜热碱性蛋白酶（TAP）会分解乳及乳制品中的蛋白质，导致乳及乳制品在储存过程中变质。因此，需要一种特定、敏感、快速且简单的方法来检测携带 TAP 的荧光假单胞菌。本研究设计了针对荧光假单胞菌 aprX 和 gyrB 基因的两组引物，优化了检测体系和条件，并建立了实时环介导等温扩增（real-time LAMP）方法，可分别在两个独立的反应管中同时检测携带 TAP 的荧光假单胞菌。以 aprX 为靶标的系统发育树表明，荧光假单胞菌和恶臭假单胞菌聚在同一分支上。以 gyrB 为靶标的系统发育树表明，荧光假单胞菌与 95%置信值的同一分支聚类，而恶臭假单胞菌聚类在不同的分支上。用实时 LAMP 检测了 16 株荧光假单胞菌和 34 株非荧光假单胞菌的 DNA。只有当 aprX 和 gyrB 的实时 LAMP 检测结果均为阳性时，才能鉴定出携带 TAP 的荧光假单胞菌。实时 LAMP 扩增产物中 aprX 和 gyrB 的解链温度分别约为 90.0°C 和 88.0°C。实时 LAMP 靶向 aprX 和 gyrB 的检测限分别为纯培养物中每反应 4.9 CFU 和脱脂乳中每反应 2.2 CFU。重复性试验的变异系数小于 2%，表明建立的荧光假单胞菌实时 LAMP 靶向 gyrB 和 aprX 具有良好的稳定性和重复性。用实时 LAMP 在 3 h 内检测了 200 份生奶样品中是否存在携带 TAP 的荧光假单胞菌，与传统方法（至少需要 5-7 天）的结果吻合率为 100%。实时 LAMP 将成为一种实用有效的方法，用于准确快速鉴定生奶中的携带 TAP 的荧光假单胞菌。
