A novel bioluminescent approach to the loop-mediated isothermal amplification-based detection of Lactobacillus salivarius in feed samples

Abstract

Coupling loop-mediated isothermal amplification (LAMP) with a bioluminescent assay in real-time (LAMP-BART) is a strategy that can be readily leveraged to detect bacteria in particular samples of interest without the need for costly or complicated equipments. However, this approach exhibits poor sensitivity, and it additionally amplifies all target DNA including that derived from non-viable cells. Herein, we sought to overcome these traditional pyrophosphate bioluminescent assay limitations by utilizing 2-deoxyadenosine-5-(α-thio) -triphosphate (dATPαS) in place of dATP when conducting LAMP, thereby markedly reducing and stabilizing overall background signal levels, resulting in a detection limit of 3 CFU/μL. We were additionally able to ouple this LAMP-BART with propidium monoazide (PMAxx™) as a means of eliminating false-positive signals derived from nonviable cells. Herein, we detail the development of this PMAxx™–LAMP–BART assay and its use for the detection of live Lactobacillus salivarius. Our developed approach exhibited 100% specificity, with a 3 CFU/μL limit of detection (LOD) pure culture. In the application of feed, the LOD was 103 CFU per 10 g of spiked dry dog food and 102 CFU per 10 g of spiked chicken feed without enrichment. Traditional culture methods and a MALDI Biotyper were also used to confirm the accuracy of our novel assay system.