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


CONCLUSIONS
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.
Yamazaki
Wataru
W
Division of Bacteriology, Osaka Prefectural Institute of Public Health, Osaka, Japan. wataru@iph.pref.osaka.jp
Ishibashi
Masanori
M
Kawahara
Ryuji
R
Inoue
Kiyoshi
K
D001427:Bacterial Toxins / Q000235:genetics; D015373:Bacterial Typing Techniques; D005516:Food Microbiology; D006460:Hemolysin Proteins / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000382:microbiology; D014735:Vibrio Infections / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C068489:thermostable direct hemolysin