期刊
JOURNAL OF FOOD PROTECTION
卷 85, 期 3, 页码 414-423出版社
INT ASSOC FOOD PROTECTION
DOI: 10.4315/JFP-21-272
关键词
aprX gene; gyrB gene; Pseudomonas fluorescens; Real-time loop-mediated isothermal amplification; Thermostable alkaline protease
资金
- National Key R&D Program of China [2018YFC1604305]
- Program of Hebei Provincial Department of Science and Technology [19222805D]
- Key Research and Development Projects of Shijiazhuang [211170212A]
In this study, a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of thermostable alkaline protease (TAP)-harboring Pseudomonas fluorescens. The method was specific, sensitive, rapid, and simple, and achieved a detection accuracy of 100% in raw milk samples.
Thermostable alkaline protease (TAP) harbored by Pseudomonasfluorescens 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 degrees 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. Realtime 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.
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