Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD)in Pyropia yezoensis.To prevent GSD from development and spread,an effective method to detect the pathogen at early GSD infection stages need to be established.In this research,PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein)and the dnaN gene(encodingβsliding clamp of DNA polymeraseⅢprotein)to detect P.marina with three primer pairs pws-dnaA2(Forward,5'-ACCGCATTAACGAACTACTCGTG-3';Reverse,5'-TGCCATTACCTACAGCATGG-3'),pcs-dnaN2(Forward,5'-CTTACAACGTTATCAGCGGC-3';Reverse,5'-GTTGAGTATTAAGTGATTGAGTAAGC-3')or pws-dnaN3(Forward,5'-ACTTACAA-CGTTATCAGCGGC-3';Reverse,5'-ACTGCTGTTTGAGTCTGCTAAC-3').Three PCR methods corresponding to the three primer pairs sufficiently distinguished P.marina from 22 bacterial species,thus resulting in detection limits of 4 to 4×10^2 CFU cells or 2.37×10^1 to 2.37×10^3 fg of P.marina DNA per PCR reaction.In an artificial infection experiment ofP.yezoensis infected with P.marina,all established PCRs successfully detected P.marina at early GSD infection stages.The results show that the established PCRs are specific and sensitive,and are potential for applications in early diagnosis of GSD in Pyropia.

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease(GSD) in Pyropia yezoensis. To prevent GSD from development and spread, an ef fective method to detect the pathogen at early GSD infection stages need to be established. In this research, PCR methods were established targeting the dnaA gene(encoding chromosome replication initiator protein) and the dnaN gene(encoding β sliding clamp of DNA polymerase Ⅲ protein) to detect P. marina with three primer pairs pws-dnaA 2(Forward, 5′-ACCGCATTAACGAACTACTCGTG-3′; Reverse, 5′-TGCCATTACCTACAGCATGG-3′), pcs-dnaN 2(Forward, 5′-CTTACAACGTTATCAGCGGC-3′; Reverse, 5′-GTTGAGTATTAAGTGATTGAGTAAGC-3′) or pws-dnaN 3(Forward, 5′-ACTTACAACGTTATCAGCGGC-3′; Reverse, 5′-ACTGCTGTTTGAGTCTGCTAAC-3′). Three PCR methods corresponding to the three primer pairs su ? ciently distinguished P. marina from 22 bacterial species, thus resulting in detection limits of 4 to 4×10 2 CFU cells or 2.37×10 1 to 2.37×10 3 fg of P. marina DNA per PCR reaction. In an arti?cial infection experiment of P. yezoensis infected with P. marina, all established PCRs successfully detected P. marina at early GSD infection stages. The results show that the established PCRs are speci?c and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.