title: Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk.
abstract: A multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) was developed and validated for simultaneous detection of Salmonella strains and Shigella strains in milk. In this system, two sets of LAMP primers were designed to specifically target invA of Salmonella spp. and ipaH of Shigella spp. Under isothermal conditions at 63 °C, ladder pattern of DNA bands could be amplified within 60 min in the presence of genomic DNAs of Salmonella strains and Shigella strains, which could be distinguished between Salmonella spp. and Shigella spp. simultaneously based on the different ladder pattern of DNA bands and subsequent restriction enzyme analysis. The overall analysis time was approximately 20 h including the enrichment of the bacterial cells, which greatly saved detection time. The sensitivity of mLAMP was found to be 100 fg DNA/tube with genomic DNAs of Salmonella strains and Shigella strains, comparatively, multiplex PCR was 1 pg DNA/tube. The mLAMP allowed the detection of milk sample artificially contaminated by Salmonella strains and Shigella strains at initial inoculation levels of approximate 5CFU/10 mL. In conclusion, the mLAMP described here can potentially facilitate simultaneous monitoring of Salmonella and Shigella in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection method.
authors.affiliation: Department of Food Safety and Microbiology, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China.
authors.forename: Yanchun
authors.initials: Y
authors.lastname: Shao
authors.forename: Shengmei
authors.initials: S
authors.lastname: Zhu
authors.forename: Cuicui
authors.initials: C
authors.lastname: Jin
authors.forename: Fusheng
authors.initials: F
authors.lastname: Chen
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology / Q000379:methods*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012760:Shigella / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial