迟钝爱德华氏菌Ⅲ型分泌系统基因的克隆及多重PCR检测方法的建立  

Cloning of type Ⅲ secretion system genes and detection of bacterium Edwardsiella tarda by multiplex PCR

作　　者：王斌[1] 赵凤梅[1] 李军伟[1] 沈皓[1] 王惠[1] 胡亮[1] 

机构地区：[1]大连海洋大学辽宁省海洋生物资源恢复与生境修复重点实验室农业部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2013年第3期217-223,共7页Journal of Dalian Ocean University

基　　金：辽宁省教育厅科学技术研究重点实验室项目(2009S026)

摘　　要：采用PCR法扩增大菱鲆Scophthalmus maximus出血性败血症病原菌迟钝爱德华氏菌Edwardsiella tardaL-49231菌株Ⅲ型分泌系统(TTSS)的装置蛋白(esaC)、效应蛋白(eseB、eseC、eseD)和调节蛋白(esrA、esrB)6种基因,并将其克隆到载体PMD19-T中分别进行测序;通过优化反应条件,建立一次检出效应蛋白eseB、eseC和eseD 3种基因片段的多重PCR方法;采用所建立的多重PCR方法检测迟钝爱德华氏菌不同菌株(L-49231、GhAn080310、GhAn080313和GhAn080314)、沙门氏菌Salmonella enterica、大肠杆菌Escherichia coli和肠杆菌Enterobacter中效应蛋白基因的存在情况。结果表明:L-49231菌株中存在esaC、eseB、eseC、eseD、esrA和esrB 6种基因片段,大小分别为837、184、934、445、464、458 bp,与GenBank中迟钝爱德华氏菌PPD130/91菌株的这6种基因的同源性分别为99%、97%、99%、99%、99%、98%;通过优化反应条件,在退火温度为54℃,基因eseB、eseC和eseD引物浓度(μmol/L)组合比为5∶10∶2,Mg2+浓度为1.5 mmol/L时,能够得到清晰、稳定且均一的多重PCR产物,并与目的条带分子量一致;采用所建立的多重PCR方法进行检测,迟钝爱德华氏菌L-49231菌株中存在eseB、eseC和eseD 3种基因片段,迟钝爱德华氏菌GhAn080310、GhAn080313、GhAn080314菌株中存在eseB和eseC基因片段,但未检出eseD基因片段;沙门氏菌、大肠杆菌和肠杆菌中均未检出eseB、eseC和eseD基因。表明所建立的多重PCR方法对迟钝爱德华氏菌具有较好的特异性,可检出具有Ⅲ型分泌系统效应蛋白的菌株。Six genes, esaC, eseB, eseC, eseD, esrA and esrB, were amplified in type Ⅲ secretion system of bacterium Edwardsiella tarda L-49231 strain isolated from infected turbot Scophthalmus maximus by PCR. Then all PCR products were cloned into vector PMD19-T and the positive clones were chosen to be sequenced. The detection method of three effector protein genes of eseB, eseC and eseD were established via the optimization of reaction conditions by multiplex PCR. The multiplex PCR detection method was then applied to determine effector protein genes of different Edwardsiella tarda strains such as L-49231, GhAn080310,GhAn080313 and GhAn080314, including Salmonella enterica, Escherichia coli and Enterobacter sp. The results showed that esaC, eseB, eseC, eseD, esrA and esrB genes were all detected in L-49231 strain, with 837, 184,934,445,464 and 458 bp in length, respectively. The homology with Edwardsiella tarda strain PPD130/91 from GenBank were found up to 99%, 97%, 99% , 99%, 99% and 98%, respectively. The optimum multi-PCR was conducted under the conditions of annealing temperature 5℃, primer concentrations（υmol/L） ratio of eseB ： eseC ： eseD = 5 ： 10 ： 2 and Mg2＋concen- tradon of 1.5 mmol/L. The eseB, eseC and eseD genes were all detected in Edwardsiella tarda strains L-49231, while only eseB and eseC genes were detected in Edwardsiella tarda strains GhAn080310, GhAn080313 and GhAn080314. But eseB, eseC and eseD genes were not detected in Salmonella enterica, Escherichia coli and Enterobacter sp. The findingds indicate that our approach of multi-PCR allows specific detection of strains with the type Ⅲ secretion system.

关 键 词：迟钝爱德华氏菌 Ⅲ型分泌系统 克隆 多重PCR 

分 类 号：S947.9[农业科学—水产养殖]