Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification

Abstract

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 × 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. Conclusion. 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. © 2008 Yamazaki et al; licensee BioMed Central Ltd.