Establishment And Application Of Triple PCR Assay For Identification Of Streptococcus Agalactiae, Streptococcus Iniae And Pseudomonas Fluoroscens

Streptococcus agalactiae is a pathogen of human, animal and fish worldwide which infectsmany aquatic animals,causes fish sepsis and meningitis, and leads to the occurrence of fulminant disease with high morbidity and mortality, and the economic losses of the aquaculture industry are severe. Streptococcus iniae is an important pathogen, which can cause fish invasive infections and outbreakof disease worldwide. Pseudomonas fluorescens is the main pathogens of red skin disease of freshwater fish, it has a wide phathogeny, can cause the outbreak of the most freshwater fish red skin disease caused huge economic losses to the freshwater fish farming. The three pathogens of aquatic animal often cause a variety of co-host infect, including the tilapia, rainbow trout, carp and so on. So it is difficult to distinguish between diagnosis only by its clinical symptoms and traditional diagnostic methods.scholors at home and aborad have studies the PCR detection method on these three bacteria, but it only limited to a one or two bacteria single or double PCR method, three aquatic animal pathogens triple PCR detection method has not yet been reported.A triple PCR method for rapid detection of Streptococcus agalactiae, Streptococcus iniae and Pseudomonas fluoroscens was established. Three pairs of specific primers based on SIP gene, LctO gene and16SPSEfluF/16SPSER gene published in GenBank were designed respectively to perform PCR with optimized of reaction system and reaction condition. The results show that, three specific bands, SIP、LctO and16SPSE were detected in the single and mixed S. agalactia,S.iniae and PFluoroscensisolates samples, and none was amplified from Streptococcusiniae,Staphylococcusaureus, Edward siellaictaluri, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas caviae or Stenotrop homonasmal tophilia. The detection limit of the S. agalactiae genomic DNA was6.557×10-2ng/mL. The triple PCR detection and the bacterial isolation and identification were performed with bacteria DNA form the tissues of liver and kidney of40tilapias challenged with S. agalactiae, S. iniae and P. fluoroscensm, the positive results by the triple PCR were consistent with the one by the bacterial isolation and identification. In conclusion, the established triple PCR in the present study was specific, sensitive, rapid and simple for the epidemiological surveillance of S. agalactiae, S. iniae and P. fluoroscens infection intilapia and other fish.