用16S rRNA基因的内切酶图谱快速鉴别几种对虾病原菌

RAPID IDENTIFICATION OF SHRIMP BACTERIAL PATHOGENS BY ENZYMATIC AMPLIFICATION AND DIGESTION OF 16S rRNA GENE

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AluI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。

Vibriosis is an important bacterial pathogen in the shrimp cultureindustry. Classical techniques of species (strain) identification are hampered by timeconsuming and complicated test protocols. Based on the 16S rRNA sequence data inthe European Molecular Biology Laboratory (EMBL), polymerase chain reachon (PCR)primers were designed and synthesized in 1994. The reference strains were Vibriocambellii (isolated and identified by the Microbiology Laboratory, Ocean University ofcyngdao in 1989-1990 from shrimp farm near Qingdao), V. paraheamolyticus and V.alginolyticus (provided by the Microbiology Research InstitUte, Chinese Academy ofSciences in September 1994), and the database accession number were X56575,X56580 and X56576, respechvely. Paired primers PL1 / PL2 were selected for afeasibility study of using them to distinguish 3 test Vibrio species, V.cambellii, Vparaheamolylicus and V.alginolyticus, by PCR and restrichon fragment lengthPolymorphisms (RFLP). PLl covered the sequence from position 166 to position 189and PL2 position 1385 to position 1409 (Escherichia coli numbering). The length ofthe arnplified fragment was 1220 base pairs.Fifty microliters amplification reachon volumes were established. After 30 cycles ofthe following incubationt:1 min at 94℃，1min at 55℃and 1.5min at 72℃, 5μl ofthe reaction mixture was used to eshmate the reachon efficiency on 1% agarose gel.The rest was purifled and digested with 2 unitS of Alu 1 restriction enLzyme. Therestrichon fragmentS were resolved on 2.5% agarose gel.The DNA fragments, asepected, can be amplifled from V. ambellii; V.parahaemo-lyticus and U.alginolyticus DNA extractS in PCR employing PL1 and PL2. Digested byenzyme AluI, the PCR products of those three bacterial species produced three differentprofiles of fragment length. Each species could be rapidly idenhified on the baois oftheir respechve band pattern. The combined uses of PCR and RFLP appear to be apowerful tool for bacterial pathogen identification in shrimp aquaculture. (Plate 1: 1,2)