Abstract
BackgroundVibrio 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.ResultsThe 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.ConclusionThe 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.
Highlights
Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders
The PCR assay required more than 4 h, while the loop-mediated isothermal amplification (LAMP) assay was markedly faster, requiring for amplification 12–18 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar from each of 34 Cholera toxin (CT)-producing V. cholerae strains and less than 45 min with spiked human feces (Fig. 1)
The assay required less than 35 min and 70 min for detection of CT-producing V. cholerae with a colony on TCBS agar and with spiked human feces from the beginning of DNA extraction to final determination
Summary
Vibrio cholerae is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Detection of CT-producing V. cholerae using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. 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. Cholera toxin (CT) is a major virulence determinant of V. cholerae. This bacterium is indigenous to fresh and blackish water environments in tropical, subtropical and temperate areas worldwide, the threat of epidemic cholera is restricted primarily to developing countries with warm climates [1,2,3]. V. cholerae causes seafood borne infection, water-borne outbreaks and epidemics in terrestrial environments [1,3].
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