title: 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 (Tmvalues) 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.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Ningwei
authors.initials: N
authors.lastname: Liu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Dayang
authors.initials: D
authors.lastname: Zou
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Derong
authors.initials: D
authors.lastname: Dong
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Da
authors.initials: D
authors.lastname: Ao
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, China.
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang
mesh_terms: D005516:Food Microbiology / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013696:Temperature; D013995:Time; D044366:Transition Temperature; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*