J Vet Sci.  2014 Mar;15(1):163-166. 10.4142/jvs.2014.15.1.163.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

• Park SB
• Kwon K
• Cha IS
• Jang HB
• Nho SW
• Fagutao FF
• Kim YK
• Yu JE
• Jung TS

• KMID: 1737623
• DOI: http://doi.org/10.4142/jvs.2014.15.1.163

Abstract

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

Keyword

Edwardsiella tarda; multiplex PCR; olive flounder; Streptococcus iniae; Streptococcus parauberis

MeSH Terms

Animals
Edwardsiella tarda/genetics/*isolation & purification
Enterobacteriaceae Infections/diagnosis/microbiology/*veterinary
Fish Diseases/*diagnosis/microbiology
Fisheries/*methods
*Flatfishes
Multiplex Polymerase Chain Reaction/economics/*veterinary
Sensitivity and Specificity
Streptococcal Infections/diagnosis/microbiology/*veterinary
Streptococcus/genetics/*isolation & purification

Figure

• 

  Fig. 1 Sensitivity of the multiplex PCR for detecting Edwardsiella (E.) tarda (415 bp), Streptococcus (S.) parauberis (718 bp), and S. iniae (300 bp). (A) Lane M, 100 bp DNA ladder; Lane 1, E. tarda; Lane 2, S. parauberis; Lane 3, S. iniae; Lane 4, E. tarda and S. parauberis; Lane 5, E. tarda and S. iniae; Lane 6, S. parauberis and S. iniae; and Lane 7, E. tarda, S. parauberis and S. iniae. (B) The multiplex PCR detected 0.01, 1, and 0.1 ng of E. tarda, S. iniae, and S. parauberis genomic DNA, respectively.

• 

  Fig. 2 Specificity of the multiplex PCR. Lane M, 100 bp DNA ladder; Lane A, E. tarda, S. parauberis, and S. iniae; Lane B, S. iniae; Lane C, S. parauberis; Lane D, E. tarda; Lane F, Lactococcus garivieae; Lane G, Aeromonas hydrophila; Lane H, Aeromonas salmonicida; Lane I, Vibrio alginolyticus; Lane J, Vibrio anguillarum; Lane K, E. hoshinae; and Lane L, E. ictaluri.

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