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Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus.

Yamazaki W, Ishibashi M, Kawahara R, Inoue K - BMC Microbiol. (2008)

Bottom Line: Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus.Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH.The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan. wataru@iph.pref.osaka.jp

ABSTRACT

Background: Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-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 Vibrio parahaemolyticus.

Results: The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 x 10(2) CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13-22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.

Conclusion: The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

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Related in: MedlinePlus

Sensitivity test for detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies [2.01 to 2.0-1 CFU per reaction]. (a) Detection of V. parahaemolyticus in pure cultures; (b) detection of V. parahaemolyticus in spiked shrimp samples.
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Figure 1: Sensitivity test for detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies [2.01 to 2.0-1 CFU per reaction]. (a) Detection of V. parahaemolyticus in pure cultures; (b) detection of V. parahaemolyticus in spiked shrimp samples.

Mentions: LAMP products were detected from all 143 V. parahaemolyticus strains. No LAMP products were detected from any of the 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains (Table 1). The PCR assay required more than 4 h, while the LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples (Fig. 1). The assay required less than 40 min and 60 min for detection of V. parahaemolyticus in a colony on TCBS agar and in spiked shrimp samples from the beginning of DNA extraction to final determination.


Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus.

Yamazaki W, Ishibashi M, Kawahara R, Inoue K - BMC Microbiol. (2008)

Sensitivity test for detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies [2.01 to 2.0-1 CFU per reaction]. (a) Detection of V. parahaemolyticus in pure cultures; (b) detection of V. parahaemolyticus in spiked shrimp samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572065&req=5

Figure 1: Sensitivity test for detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies [2.01 to 2.0-1 CFU per reaction]. (a) Detection of V. parahaemolyticus in pure cultures; (b) detection of V. parahaemolyticus in spiked shrimp samples.
Mentions: LAMP products were detected from all 143 V. parahaemolyticus strains. No LAMP products were detected from any of the 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains (Table 1). The PCR assay required more than 4 h, while the LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples (Fig. 1). The assay required less than 40 min and 60 min for detection of V. parahaemolyticus in a colony on TCBS agar and in spiked shrimp samples from the beginning of DNA extraction to final determination.

Bottom Line: Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus.Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH.The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan. wataru@iph.pref.osaka.jp

ABSTRACT

Background: Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-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 Vibrio parahaemolyticus.

Results: The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 x 10(2) CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13-22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.

Conclusion: The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

Show MeSH
Related in: MedlinePlus