AUTHOR=Zhang Shufei , Hu Lianxia , Xue Yuling , Zhang Dong , Zhang Yaoguang , Wang Shijie 

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

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1133077

DOI=10.3389/fmicb.2023.1133077

ISSN=1664-302X

ABSTRACT=<sec><title>Introduction</title><p>The psychrophilic bacterium <italic>Pseudomonas lurida</italic> (<italic>P. lurida</italic>) and its thermostable alkaline proteases can seriously damage raw milk quality.</p></sec><sec><title>Methods</title><p>In this study, specific primers were designed for <italic>P. lurida’s gyrB</italic> and <italic>aprX</italic> genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of <italic>P. lurida</italic> and its proteases in raw milk. A phylogenetic tree of the <italic>gyrB</italic> and <italic>aprX</italic> genes of <italic>P. lurida</italic> was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of <italic>P. lurida</italic> and 44 strains of non-<italic>P. lurida</italic> were detected <italic>via</italic> RealAmp to analyze the specificity of the primer.</p></sec><sec><title>Results</title><p>It was found that <italic>aprX</italic>-positive proteases were produced by <italic>P. lurida-</italic>positive strains only when <italic>Pseudomonas fluorescens</italic> was negative. The dissociation temperatures of <italic>gyrB</italic> and <italic>aprX</italic> 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 <italic>P. lurida</italic> in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of <italic>P. lurida DNA</italic> 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 <italic>P. lurida</italic> was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of <italic>P. lurida</italic> and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of <italic>P. lurida</italic> to produce proteases reached 88%.</p></sec><sec><title>Discussion</title><p>In conclusion, RealAmp established an early and rapid method for the detection of <italic>P. lurida</italic> 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.</p></sec>