title: Development of a LAMP assay for rapid detection of different intimin variants of attaching and effacing microbial pathogens.
abstract: Intimin harboured by pathogenic Escherichia coli (E. coli) strains is a key virulence factor involved in host cell adherence and colonization. Twenty-seven intimin-encoding E. coli attaching and effacing (eae) gene variants have been reported according to their 3' binding domain sequences. In our study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect all known intimin variants. Four primers specific for six regions of eae genes were designed using online software. The eae-LAMP assay was highly specific and detected all 27 tested eae variants; no cross-reactions were observed with genes from enterotoxigenic E. coli (ETEC), E. coli BL21, Salmonella, Shigella, Listeria monocytogenes, or Streptococcus suis type 2 (SS2). With the lowest detection limit of approximately 10 copies per reaction the eae-LAMP assay was 100 times more sensitive than conventional PCR. These results, and the results of tests involving food and faecal samples artificially contaminated with E. coli O157 : H7 (eaeγ+), show that the eae-LAMP assay is a simple, rapid, sensitive and specific tool for detecting intimin variants from pathogenic strains of E. coli. The eae-LAMP assay has great potential for wider applications, not only in the laboratory but also in the field setting, as it does not require specialized equipment.
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences; Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry; National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Xue-Han
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences; Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry; National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Qing
authors.affiliation: Nanjing Tech-Bank Bio-industry Co. Ltd, Nanjing 210000, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Ya-Dong
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences; Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry; National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: Li
authors.initials: L
authors.lastname: Bin
authors.affiliation: Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Ivanek
authors.initials: I
authors.lastname: Renata
authors.affiliation: Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences; Key Laboratory of Engineering Research of Veterinary Bio-products of Agricultural Ministry; National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, China.
authors.forename: He
authors.initials: H
authors.lastname: Kong-Wang
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics*; D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary; D029968:Escherichia coli Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D018829:Adhesins, Bacterial; D017931:DNA Primers; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli