Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification.
BACKGROUND
Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of V. cholerae. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing Vibrio cholerae.


RESULTS
The assay provided markedly more sensitive and rapid detection of CT-producing V. cholerae strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing V. cholerae strains, but did not detect 13 CT non-producing V. cholerae and 53 non-V. cholerae strains. Sensitivity of the LAMP assay for direct detection of CT-producing V. cholerae in spiked human feces was 7.8 x 102 CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing V. cholerae required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination.


CONCLUSIONS
The LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing V. cholerae and will be useful in facilitating the early diagnosis of human V. cholerae infection.
Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan. wataru@iph.pref.osaka.jp
Wataru
W
Yamazaki
Kazuko
K
Seto
Masumi
M
Taguchi
Masanori
M
Ishibashi
Kiyoshi
K
Inoue
D015373:Bacterial Typing Techniques; D001483:Base Sequence; D002771:Cholera / Q000382:microbiology*; D002772:Cholera Toxin / Q000235:genetics* / Q000378:metabolism; D005243:Feces / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D002772:Cholera Toxin