Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification.
A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli.
Hara-Kudo
Yukiko
Y
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Nemoto
Jiro
J
Eiken Chemical Co. Ltd, 143 Nogi Nogimachi, Shimotsuga-gun, Tochigi 329-0114, Japan.
Ohtsuka
Kayoko
K
Saitama Institute of Public Health, Saitama 338-0824, Japan.
Segawa
Yuko
Y
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Takatori
Kosuke
K
Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Kojima
Tadashi
T
Eiken Chemical Co. Ltd, 143 Nogi Nogimachi, Shimotsuga-gun, Tochigi 329-0114, Japan.
Ikedo
Masanari
M
Eiken Chemical Co. Ltd, 143 Nogi Nogimachi, Shimotsuga-gun, Tochigi 329-0114, Japan.
D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics*; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D031224:Raphanus / Q000382:microbiology; D012680:Sensitivity and Specificity; D022603:Shiga Toxins / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022603:Shiga Toxins