Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

Abstract

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 10 cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10 cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10 cfu/reaction, which was 10 lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.

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

“…For P. fluorescens in pure culture and pasteurized milk, the detection limits of the LAMP assay were 2.57 × 10 2 and 3 × 10 2 CFU/mL, respectively, which were applicable for the detection of P. fluorescens in raw milk during the actual production process. It was worth mentioning that, compared with the previous study (Xin et al, 2017), the addition of loop primers shortened the detection time to 30 min and improved the efficiency significantly.…”

“…The exploitation of a specific LAMP assay for detecting P. fluorescens in raw milk has attracted much attention. In our previous study, a LAMP assay system was established based on the conserved gene sequence of P. fluorescens 38 lipases, and we evaluated its application in raw milk ( Xin et al, 2017 ). Although the reaction time of this LAMP system was 50 min, the dairy enterprise expected a faster detection speed.…”

Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food

“…Current Salmonella testing in foods relies on microbiological culturing, which consists of time-consuming and labor-intensive procedures that require days or weeks for a definitive result (International Organization for Standardization [ISO], 2017; United States Department of Agriculture [USDA], 2017; Andrews et al, 2018). Rapid, sensitive, and specific nucleic acid amplification tests (NAATs), including PCR, real-time quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), have been developed and applied in the detection and identification of Salmonella in foods (Malorny et al, 2009; Balachandran et al, 2012; Lofstrom and Hoorfar, 2012; Cheng et al, 2015; Yang et al, 2016; Domesle et al, 2018; Hu et al, 2018). The isothermal LAMP method, in particular, has gained wide popularity as highlighted in a recent comprehensive review (Yang et al, 2018).…”

Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection

“…In one of the studies, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the P. fluorescens in raw milk (cow), as most of the frequently reported heat-resistant lipase-producing bacterial species with detection limit of 4.8 × 10 2 cfu/reaction of the template DNA and 7.4 × 10 1 cfu/reaction of P. fluorescens led to contamination of pasteurized cow milk (Xin et al 2017). LAMP assay cannot distinguish between DNA from viable cells and to that from dead cells (Chen et al 2011;Wan et al 2012).…”