AI Article Synopsis
- The study discusses an innovative LAMP-BART method to detect live bacteria, combining loop-mediated isothermal amplification with a bioluminescent assay while overcoming limitations of traditional techniques.
- By substituting dATP with dATPαS, the new method increases sensitivity and reduces background noise, allowing detection limits as low as 3 CFU/μL.
- The PMAxx™ approach eliminates false positives from nonviable cells, achieving 100% specificity, with effective applications in detecting Lactobacillus salivarius in various sample types, including dog and chicken feed.
Article 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 10 CFU per 10 g of spiked dry dog food and 10 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.
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| http://dx.doi.org/10.1016/j.mimet.2021.106209 | DOI Listing |
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