Development of a real-time loop-mediated isothermal amplification assay for the sensitive and rapid detection of Listeria monocytogenes

Abstract

Conventional culture and molecular detection methods are always time consuming and require a specific laboratory infrastructure, thereby restricting their use for the rapid detection and diagnosis of pathogens. A real-time loop-mediated isothermal amplification (RealAmp) assay performed by ESEtube scanner to rapidly detect Listeria monocytogenes isolated from food was developed. The results showed that the RealAmp assay using the tube scanner was more efficient and precise than the conventional API Listeria assay and the real-time PCR assay.

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

“…The LAMP assay was revealed to have a similar limit of detection in pasteurized cow milk and pure culture. Comparing this to the existing literature, 2 studies have previously reported that the results produced from the LAMP assay in detecting P. fluorescens in pure culture and in artificially contaminated milk samples were of a similar order of magnitude (Cho et al, 2014;Ye et al, 2015). Three out of 5 published studies have suggested that the detection limit of the method is lower in pure culture than in milk (Yang et al, 2011;Wang et al, 2015;Cornelissen et al, 2016).…”

“…Portable fluorescence detectors have already been applied to on-site detection of microorganisms and have been shown to deliver the same results as fluorescence quantification PCR instruments (Fu et al, 2016). Moreover, a battery-operated portable fluorescence detector can be used in conjunction with the LAMP assay system (Ye et al, 2015;Lee et al, 2016). Furthermore, because the LAMP reaction tubes are not opened until the reaction has completed, the potential risks of cross-contamination and false- ; lanes 1, 2, 3, 4, 5, 6, and 7 represent 4.8 × 10 5 , 4.8 × 10 4 , 4.8 × 10 3 , 4.8 × 10 2 , 4.8 × 10 1 , 4.8 × 10 0 , and 4.8 × 10 −1 cfu/reaction, respectively.…”

Rapid and Specific Detection of Listeria monocytogenes With an Isothermal Amplification and Lateral Flow Strip Combined Method That Eliminates False-Positive Signals From Primer–Dimers

“…It was highly specific to L. monocytogenes and was able to detect as low as 1 CFU of the bacterium per reaction (50 µl) without DNA purification, or 100 fg of the genomic DNA/50 µl. This sensitivity was higher than the LAMP testing limit of 10 3 CFU/ml and was comparable to the detection limit of PCR-or qPCR-based methods, which was between 10 2 and 10 3 CFU/ml (Ye et al, 2015;Jayanth and Varadaraj, 2017;Garrido-Maestu et al, 2018). The amplification could be conducted under the temperature between 37 and 42 • C, and the whole detection finished within 25 min.…”

Review in isothermal amplification technology in food microbiological detection

“…Compared with real-time PCR method, RealAmp has the improved specificity and sensitivity. Ye et al ( 2015 ) compared RealAmp with the API Listeria and real-time PCR assays to detect Listeria . All 58 L. monocytogenes strains from different countries were detected by the RealAmp assay, whereas only 57 (98.3%) and 56 (96.6%) strains were identified as L. monocytogenes by the API Listeria and PCR assays respectively, thereby demonstrating that it has a higher specificity and sensitivity than conventional identification assays.…”