Cite this paper:
YANG Huichao, YAN Yongwei, LI Jie, TANG Lei, MAO Yunxiang, MO Zhaolan. Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis[J]. Journal of Oceanology and Limnology, 2020, 38(1): 168-176

Development of a PCR method for detection of Pseudoalteromonas marina associated with green spot disease in Pyropia yezoensis
YANG Huichao1,2, YAN Yongwei1, LI Jie1, TANG Lei3, MAO Yunxiang3, MO Zhaolan1,2
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 Affairs, 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
Abstract:
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 (encoding β sliding clamp of DNA polymerase Ⅲ protein) 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'-ACTTACAACGTTATCAGCGGC-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 4×102 CFU cells or 2.37×101 to 2.37×103 fg of P. marina DNA per PCR reaction. In an artificial 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 specific and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.
Key words:    Pyropia yezoensis|green spot disease (GSD)|Pseudoalteromonas marina|PCR detection|early diagnosis   
Received: 2019-02-27   Revised:
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