Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture

Shufei Zhang; Lianxia Hu; Yuling Xue; Dong Zhang; Yaoguang Zhang; Shijie Wang

doi:10.3389/fmicb.2023.1133077

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture

Front Microbiol. 2023 Apr 12:14:1133077. doi: 10.3389/fmicb.2023.1133077. eCollection 2023.

Authors

Shufei Zhang¹, Lianxia Hu², Yuling Xue³, Dong Zhang³, Yaoguang Zhang³, Shijie Wang^{1

3}

Affiliations

¹ College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang, Hebei, China.
² College of Chemical Engineering, Shijiazhuang University, Shijiazhuang, Hebei, China.
³ Junlebao Dairy Group Co., Ltd., Shijiazhuang, Hebei, China.

Abstract

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.

Keywords: Pseudomonas lurida; aprX gene; gyrB gene; raw milk; real-time LAMP; thermostable alkaline proteases.