Open AccessDevelopment and evaluation of a multiplex PCR assay for simultaneous detection ofFlavobacterium psychrophilum,Yersinia ruckeriandAeromonas salmonicidasubsp.salmonicidain culture fisheries
Author(s) -
Ertan Emek Onuk,
Alper Çiftçi,
Arzu Fındık,
Yüksel Durmaz
Publication year - 2010
Publication title -
journal of veterinary science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 47
eISSN - 1976-555X
pISSN - 1229-845X
DOI - 10.4142/jvs.2010.11.3.235
Subject(s) - aeromonas salmonicida , yersinia ruckeri , microbiology and biotechnology , biology , multiplex polymerase chain reaction , polymerase chain reaction , multiplex , bacteria , primer (cosmetics) , serial dilution , virology , fish <actinopterygii> , gene , chemistry , fishery , medicine , bioinformatics , biochemistry , genetics , alternative medicine , organic chemistry , pathology , rainbow trout
Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.