Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus

Abstract

Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry.

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

“…This indicated Bst 3.0 could be a useful model for studying non-specific amplification. We observed that early amplifying products corresponded to specific amplification events, and the later products corresponded to non-specific amplification, supporting our prediction that we could use T m as a proxy for sequence identity, as is common with PCR and has been used previously in LAMP (25)(26)(27)(28)(29).…”

Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenicLeptospira

“…2b). Similar melt curve-based real-time LAMP analysis was reported for clinical pathogens like Salmonella and Vibrio (Liu et al 2017), Arcanobacterium pluranimalium (Abdulmawjood et al 2015) and Herpes virus (Chen et al 2013). The melting curve analysis was used for species level genotyping of pathogenic Leptospira by targeting variable-number tandem repeats (Naze et al 2015).…”

“…Similar melt curve‐based real‐time LAMP analysis was reported for clinical pathogens like Salmonella and Vibrio (Liu et al . ), Arcanobacterium pluranimalium (Abdulmawjood et al . ) and Herpes virus (Chen et al .…”

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…17,31 LAMP has the capacity for incorporation into assays for medical investigation, genetic testing, environmental testing and rapid testing of food products, especially in resource-constrained environments. 17 LAMP has found application in detecting resistance genes such as bla KPC and bla NDM-1 in E. coli, Klebsiella pneumonia and Acinetobacter baumannii 32 as well as identification of bacteria like E. coli, 33 Mycobacteria, 34 Vibrio parahemolyticus, Salmonella spp 35 and A. baumannii. 36 LAMP with a detection limit of 1ng has also been successfully used for rapid detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, both of which cause periodontitis in humans.…”