Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk

Abstract

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

“…The purified CQMED Bst -LF has very similar activity to that previously reported for the standard Bst 2.0 WarmStart DNA Polymerase (NEB), in the concentration range of 8–12 ng/µL. 52,53 We did not observe a significant difference between the produced HIV -RT and the standard commercial enzymes tested, using a protocol of 10–30 min reaction at 50 °C–55 °C. As the reverse transcriptase activity can vary according to the RT type, template sequence, or reaction conditions, 54,55 it would be important to further optimize the enzyme for difficult templates, especially rich in CG sequences, in case the enzyme is used in other applications.…”

Single Molecule Real-Time Sequencing and Traditional Cultivation Techniques Reveal Complex Community Structures and Regional Variations of Psychrotrophic Bacteria in Raw Milk

“…These organisms also secrete heat-resistant proteases and lipases that can significantly alter the quality of dairy products. For example, lipid hydrolysis by contaminating lipases can lead to rancidity while released proteases hydrolyze casein and produces peculiar smells and cause aging and gelation thereby shortening the shelf life ( Xin et al, 2017 ; Meng et al, 2018 ; Yang et al, 2020 ; Bu et al, 2022 ).…”