Abstract
Vibrio cholerae and Vibrio vulnificus are critical foodborne pathogens that need to be intensively controlled for their infection due to the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for detecting Vibrio spp. is thiosulfate-citrate-bile salts-sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem with processing PCR two times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria–Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.
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Acknowledgements
The author acknowledges Dokyun Oh for her technical help during the experiment.
Funding
This research was also a part of the project titled 'Development of on-site diagnosis system for microbiological hazards in the fishery environment, funded by the Ministry of Oceans and Fisheries, Korea. This research was also supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education (2021R1A6A1A03039211).This study was also supported by the grant (No. 18162MFDS542 in 2018 and 2019) from the Ministry of Food and Drug Safety of Korea.
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JHL and YMK designed and lead the writing and analysis of data. JHL, MGK, DHL, and DMJ were performed in the sampling, experiment, and analyzed data. JHL, SKP, FK, and YMK were involved in the data analysis, writing, and editing of the manuscript.
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Jae-Hwa Lee, Seul-Ki Park, Fazlurrahman Khan, Du-Min Jo, Do-ha Lee, Min-Gyun Kang, and Young-Mog Kim declare that they have no conflict of interest.
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Lee, JH., Park, SK., Khan, F. et al. Simultaneous isolation and enumeration of virulent Vibrio cholerae and Vibrio vulnificus using an advanced MPN-PCR method. Arch Microbiol 204, 5 (2022). https://doi.org/10.1007/s00203-021-02613-y
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DOI: https://doi.org/10.1007/s00203-021-02613-y