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

作　　者：YANG Huichao YAN Yongwei LI Jie TANG Lei MAO Yunxiang MO Zhaolan 

机构地区：[1]Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Aff airs,Qingdao Key Laboratory of Mariculture,Epidemiology and Biosecurity,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China [2]College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China [3]Laboratory for Marine Biology and Biotechnology,Qingdao National Laboratory for Marine Science and Technology,Key Laboratory of Marine Genetics and Breeding(Ocean University of China),Ministry of Education,Qingdao 266003,China

出　　处：《Journal of Oceanology and Limnology》2020年第1期168-176,I0012-I0015,共13页海洋湖沼学报（英文）

基　　金：Supported by the China Agriculture Research System(No.CARS-50);the National High-Tech R&D Program of China(No.2012AA10A406);the National Science and Technology Infrastructure Platform Construction(No.2018DKA30470);the Aoshan Technology Innovation Program of Qingdao National Laboratory for Marine Science and Technology(No.2015ASKJ02)

摘　　要：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.

关 键 词：Pyropia YEZOENSIS green SPOT disease (GSD) PSEUDOALTEROMONAS MARINA PCR DETECTION EARLY diagnosis 

分 类 号：R73[医药卫生—肿瘤]