Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus.
OBJECTIVE
To develop a haemolysin (hly) gene-based species-specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus.


RESULTS
The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species-specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube.


CONCLUSIONS
Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species.


CONCLUSIONS
Because there is lack of simple, rapid and cost-effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.
Haldar
S
S
Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531, Japan.
Neogi
S B
SB
Kogure
K
K
Chatterjee
S
S
Chowdhury
N
N
Hinenoya
A
A
Asakura
M
M
Yamasaki
S
S
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D006460:Hemolysin Proteins / Q000235:genetics*; D033561:Penaeidae; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000235:genetics / Q000302:isolation & purification; D014735:Vibrio Infections / Q000175:diagnosis / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; C073845:hemolysin, Vibrio