The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes

Abstract

Abstract:Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63˝C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Lab-Made Electronic Nose for Fast Detection of Listeria monocytogenes and Bacillus cereus

“…ELISA, on the other hand, requires sample enrichment and processing before analysis, has instability of antibodies, and a risk of false positive/negative [12]. An electronic nose (e-nose), on the other hand, has been reported as successful in differentiating different samples according to organic volatile compounds (VOCs) emanated from the samples [13,14]. Recently, e-noses are widely used for analysis in many fields of science and industry (e.g., medicine, safety, the food industry, pharmaceuticals, and the chemical and environmental protection industries) [15].…”

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

“…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). The DNA extraction method used is an important factor that may affect the results obtained (Sowmya et al, 2012).…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…[Colour figure can be viewed at wileyonlinelibrary.com] instance, Salmonella typhi (Abdullah et al 2014;Fan et al 2015), Campylobacter jejuni and Campylobacter coli (Pham et al 2015), and Helicobacter pylori (Bakhtiari et al 2016;Yari et al 2016). Other successful examples of LAMP usage include Neisseria meningitidis (Lee et al 2015), Listeria monocytogenes (Birmpa et al 2015;Wang et al 2015b;Ye et al 2015), Entamoeba histolytica (Rivera and Ong 2013;Singh et al 2013;Mwendwa et al 2017), and human leptospirosis (Seesom et al 2015;Hsu et al 2017). LAMP was also efficiently formulated against several multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii (Kim et al 2016b).…”