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
| Autor: | Hiroko Yamada, Tetsuya Mori, Atsuko Tanouchi, Yuhki Nagai, Junko Isobe, Hiroshi Nakagawa, Fumie Suzuki, Kaori Iwabuchi, Kayoko Ohtsuka, Rie Kikuchi, Yasufumi Ueda, Noriko Konishi, Takumiko Yamazaki, Yukiko Hara-Kudo, Jun Terajima |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
food.ingredient Meat 030106 microbiology Food Contamination Biology medicine.disease_cause Immunomagnetic separation Escherichia coli O157 Real-Time Polymerase Chain Reaction Serogroup Microbiology Raphanus 03 medical and health sciences food medicine Agar Animals Escherichia coli Shiga toxin-producing Escherichia coli Chromogenic Inoculation Immunomagnetic Separation O Antigens Shiga toxin General Medicine Molecular Typing 030104 developmental biology Real-time polymerase chain reaction biology.protein Food Microbiology Cattle Food Science |
| Zdroj: | International journal of food microbiology. 230 |
| ISSN: | 1879-3460 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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