| 摘要: |
| 坎普氏弧菌是青岛海洋大学生物系微生物实验室于1989－1990年自对虾养殖场中国对虾红腿病虾心脏及血淋巴中分离并鉴定的菌株，副溶血弧菌和溶藻胶弧菌两菌株于1994年9月得自中国科学院微生物研究所。为建立快速、准确的中国对虾病原菌的诊断技术，根据几种细菌的16SrRNA基因的序列，设计并合成该基因的多聚酶链反应的引物PLI和PL2。用该对引物分别从坎普氏弧菌、副溶血弧菌和溶藻胶弧菌的DNA样品中扩增出分子量与原设计相同的DNA片段，然后用Alu I内切酶对3种细菌的多聚酶链反应产物进行酶切，形成3种不同的限制性内切酶图谱。研究结果证明，用16SrRNA基因的限制性内切酶图谱，可快速、准确地鉴别3种对虾病原菌。 |
| 关键词: 弧菌 16SrRNA 多聚酶链反应 限制性内切酶图谱 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目，39370545号 |
附件 |
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| RAPID IDENTIFICATION OF SHRIMP BACTERIAL PATHOGENS BY ENZYMATIC AMPLIFICATION AND DIGESTION OF 16S rRNA GENE |
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Kong Jie1, Liu Ping1, Zhang Yan1, Yang Conghai1, Ye Jun2, Xu Huishu2, Guo Huarong3
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1.Yellow Sea Fisheries Aisearch Institute, Chinese Academy of Fishery Sciences， Qingdao 266003;2.Department of Biology , Qingdao Ocean University, Qingdao 266003;3.Institute of Oceanology , Chinese Academy of Sciences, Qingdao 266071
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| Abstract: |
| Vibriosis is an important bacterial pathogen in the shrimp culture industry. Classical techniques of species (strain) identification are hampered by time consuming and complicated test protocols. Based on the 16S rRNA sequence data in the European Molecular Biology Laboratory (EMBL), polymerase chain reaction (PCR) primers were designed and synthesized in 1994. The reference strains were Vibriu cambellii (isolated and identi?ed by the Microbiology Laboratory, Ocean University of Qingdao in 1989 - 1990 from shrimp farm near Qingdao), V. paraheamoticus and V. alginolyticus (provided by the Microbiology Research Institute, Chinese Academy of Sciences in September 1994), and the database accession numbers are X56575, X56580 and X56576, respectively. Paired primers PL1/ PL2 were selected for a feasibility study of using them to distinguish 3 test Vibrio species, V. cambellii, V. paraheamolyticus and V. alginolyticus, by PCR and restriction fragment length polymorphisms (RFLP). PLl covered the sequence from position 166 to position 189 and PL2 position 1385 to position 1409 (Escherichia coli numbering). The length of the amplified fragment was 1220 base pairs.
Fifty microliters amplification reaction volumes were established. After 30 cycles of the following incubation: 1 min at 94°C, l min at 55°C and l.5min at 72°C, 5μl of the reaction mixture was used to estimate the reaction efficiency on 1% agarose gel. The rest was purified and digested with 2 units of Alu I restriction enzyme. The restriction fragments were resolved on 2.5% agarose gel.
The DNA fragments, as expected, can be amplified from V. cambellii, V. parahaemolyticus and V. alginolyticus DNA extracts in PCR employing PL1 and PL2. Digested by enzyme Alu I, the PCR products of those three bacterial species produced three different profiles of fragment length. Each species could be rapidly identified on the basis of their respective band pattern. The combined uses of PCR and RFLP appear to be a powerful tool for bacterial pathogen identification in shrimp aquaculture. (Plate I: 1, 2) |
| Key words: Vibrio, 16S rRNA, PCR, RFLP |
