title: Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli.
abstract: The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 10(4)CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6 CFU per 25 g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.
authors.affiliation: R&D Center, Nippon Meat Packers, Inc, 3-3 Midorigahara, Tsukuba, Ibaraki 300-2646, Japan. t.yonekita@nipponham.co.jp
authors.forename: Taro
authors.initials: T
authors.lastname: Yonekita
authors.forename: Ryuji
authors.initials: R
authors.lastname: Ohtsuki
authors.forename: Eri
authors.initials: E
authors.lastname: Hojo
authors.forename: Naoki
authors.initials: N
authors.lastname: Morishita
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.forename: Fumiki
authors.initials: F
authors.lastname: Morimatsu
mesh_terms: D000906:Antibodies; D023181:Antimicrobial Cationic Peptides; D001431:Bacteriological Techniques / Q000379:methods*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D054323:Shiga-Toxigenic Escherichia coli / Q000302:isolation & purification*
chemical_list: D000906:Antibodies; D023181:Antimicrobial Cationic Peptides