title: An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.
abstract: To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups.
authors.affiliation: Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. Electronic address: ykudo@nihs.go.jp.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Hara-Kudo
authors.affiliation: Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku, Tokyo 169-0073, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Konishi
authors.affiliation: Saitama Institute of Public Health, 410-1, Ewai, Yoshimi-machi, Hiki-gun, Saitama 355-0133, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Ohtsuka
authors.affiliation: Research Institute for Environmental Sciences and Public Health of Iwate Prefecture, 1-11-16, Kitaiioka, Morioka 020-0857, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Iwabuchi
authors.affiliation: Fukushima Institute for Public Health, 16-6, Mitouchi, Houkida, Fukushima 960-8560, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Kikuchi
authors.affiliation: Toyama Institute of Health, 17-1, Nakataikouyama, Imizu 939-0363, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Isobe
authors.affiliation: Suginami City Institute of the Public Health, 3-20-3, Takaidohigashi, Suginami, Tokyo 168-0072, Japan.
authors.forename: Takumiko
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Shizuoka City Institute of Environmental Sciences and Public Health, 1-4-7, Oguro, Suruga, Shizuoka 422-8072, Japan.
authors.forename: Fumie
authors.initials: F
authors.lastname: Suzuki
authors.affiliation: Mie Prefecture Health and Environment Research Institute, 3684-11, Sakura-cho, Yokkaichi 512-1211, Japan.
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Nagai
authors.affiliation: Hiroshima Prefectural Technology Research Institute, Public Health and Environment Center, 1-6-29, Minami-machi, Minami, Hiroshima 734-0007, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yamada
authors.affiliation: Hiroshima City Institute of Public Health, 4-1-2, Shoko-Center, Nishi, Hiroshima 733-8650, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Tanouchi
authors.affiliation: Institute for Food and Environment Sciences Tokyo Kenbikyo-in Foundation, 4F, 5-1, Toyomi-cho, Chuo, Tokyo 104-0055, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mori
authors.affiliation: BML Food Science Solutions, Inc., 1491, Matoba, Kawagoe 350-1101, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakagawa
authors.affiliation: Center of Inspection of Imported Foods and Infectious Diseases, Kobe Quarantine Station, 1-1, Toyahama-cho, Hyogo, Kobe 652-0866, Japan.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Terajima
mesh_terms: D000818:Animals; D002417:Cattle; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D018189:Immunomagnetic Separation / Q000379:methods; D008460:Meat / Q000382:microbiology*; D058889:Molecular Typing / Q000379:methods*; D019081:O Antigens / Q000235:genetics*; D031224:Raphanus / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D065288:Serogroup
chemical_list: D019081:O Antigens
keywords: Detection; Interlaboratory study; Method; Non-O157; O157; Shiga toxin-producing Escherichia coli