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Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture.
Zhang, Shufei; Hu, Lianxia; Xue, Yuling; Zhang, Dong; Zhang, Yaoguang; Wang, Shijie.
Afiliación
  • Zhang S; College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang, Hebei, China.
  • Hu L; College of Chemical Engineering, Shijiazhuang University, Shijiazhuang, Hebei, China.
  • Xue Y; Junlebao Dairy Group Co., Ltd., Shijiazhuang, Hebei, China.
  • Zhang D; Junlebao Dairy Group Co., Ltd., Shijiazhuang, Hebei, China.
  • Zhang Y; Junlebao Dairy Group Co., Ltd., Shijiazhuang, Hebei, China.
  • Wang S; College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang, Hebei, China.
Front Microbiol ; 14: 1133077, 2023.
Article en En | MEDLINE | ID: mdl-37125188
Introduction: The psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality. Methods: In this study, specific primers were designed for P. lurida's gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer. Results: It was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/µL and that in the 10% skimmed milk was 5.5 copies/µL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%. Discussion: In conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Microbiol Año: 2023 Tipo del documento: Article País de afiliación: China Pais de publicación: Suiza

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Microbiol Año: 2023 Tipo del documento: Article País de afiliación: China Pais de publicación: Suiza