J Vet Sci.  2010 Sep;11(3):235-241. 10.4142/jvs.2010.11.3.235.

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Affiliations
  • 1Department of Diseases of Aquatic Animals, Faculty of Veterinary Medicine, Ondokuz Mayis University, 55139, Kurupelit, Samsun, Turkey.
  • 2Department of Microbiology, Faculty of Veterinary Medicine, Ondokuz Mayis University, 55139, Kurupelit, Samsun, Turkey. aciftci@omu.edu.tr
  • 3Veterinary Control and Research Institute, Samsun, Turkey.

Abstract

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.

Keyword

Aeromonas salmonicida subsp. salmonicida; Flavobacterium psychrophilum; multiplex PCR; Yersinia ruckeri

MeSH Terms

Aeromonas salmonicida/*genetics
Animals
DNA Primers/genetics
Fish Diseases/*diagnosis/*microbiology
Fishes
Flavobacterium/*genetics
Gram-Negative Bacterial Infections/diagnosis/*veterinary
Polymerase Chain Reaction/methods/*veterinary
Yersinia rucker/*genetics

Figure

  • Fig. 1 Simultaneous identification of Flavobacterium (F.) psychrophilum (971 bp), Yersinia (Y.) ruckeri (575 bp), and Aeromonas (A.) salmonicida subsp. salmonicida (422 bp) by m-PCR3. Lane M: Molecular size marker (100~1,000 bp), Lane 1: m-PCR with the three pathogens together, Lane 2: A. salmonicida subsp. salmonicida alone, Lane 3: Y. ruckeri alone, Lane 4: F. psychrophilum alone, Lane 5: F. psychrophilum and Y. ruckeri, Lane 6: F. psychrophilum and A. salmonicida subsp. salmonicida, Lane 7: Y. ruckeri and A. salmonicida subsp. salmonicida.

  • Fig. 2 Determination of detection limit of m-PCR assay. Lane M: Molecular size marker (100~1,000 bp), Lane a-h: the serial dilutions of chromosomal DNA extracted from reference F. psychrophilum, Y. ruckeri and A. salmonicida subsp. salmonicida strains, from left to right, 1,055, 700, 350, 210, 140, 70, 35 and 17 pg DNA, respectively.

  • Fig. 3 Detection limit of m-PCR assay in pure cultures of bacteria. Lane M: Molecular size marker (100~1,000 bp), Lane 1: 1.0 × 103, Lane 2: 500, Lane 3: 30, Lane 4: 15 CFU of F. psychrophilum, Y. ruckeri and A. salmonicida subsp. salmonicida, respectively.

  • Fig. 4 The detection limit of m-PCR assay in the presence of tissue debris. Lane M: Molecular size marker (100~1,000 bp), Lane 1: 1.0 × 105, Lane 2: 1.0 × 104, Lane 3: 1.0 × 103, Lane 4: 250 Lane 5: 125 CFU of F. psychrophilum, Y. ruckeri and A. salmonicida subsp. salmonicida, respectively.


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