同步检测海水养殖动物5种病原菌的扩增子拯救多重PCR(Arm-PCR)方法的建立与应用  

Establishment and Application of Amplicon Rescue Multiplex PCR(Arm-PCR) for the Simultaneous Detection of Five Major Bacterial Pathogens from Marine Aquaculture Animals

作　　者：耿伟光[1,2] 史成银[1] 李晋[1] 黄倢[1] 王秀华[1] 谢国驷[1] 

机构地区：[1]农业部海洋渔业可持续发展重点实验室中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《农业生物技术学报》2013年第9期1125-1134,共10页Journal of Agricultural Biotechnology

基　　金：中国水产科学研究院黄海水产研究所"中央级公益性科研院所基本科研业务费专项资金"(No.20603022012009);国家科技支撑计划(No.2012BAD17B01)

摘　　要：鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(V.harveyi)、嗜水气单胞菌(Aeromonas hydrophila)、迟缓爱德华氏菌(Edwardsiella tarda)和副溶血弧菌(V.parahaemolyticus)是我国海水养殖动物5种重要的病原菌。本研究在分析了其致病因子基因的基础上,依据扩增子拯救多重PCR(Arm-PCR)的原理,设计了5套特异性的套式PCR引物,并在各内引物的5'端分别加上能被一对通用引物识别的标签序列;通过对套式PCR中影响扩增结果的引物混合物浓度、退火温度、Mg2+浓度、dNTPs浓度以及Taq DNA聚合酶浓度等5个反应参数的调整和优化,最终建立了一种能同步检测海水养殖动物5种常见病原菌的Arm-PCR方法。优化后的Arm-PCR方法第一步PCR反应体系为:10×PCR Buffer 5μL(含20 mmol/L的Mg2+),dNTP(各2.5mmol/L)5μL,Taq酶(2.5 U/μL)0.6μL,10×Primer Mix(2μmol/L)5μL,DNA模板各1μL,灭菌双蒸馏水补足至50μL,反应的退火温度为55℃。实验结果表明:对鳗弧菌、哈维氏弧菌、嗜水气单胞菌、迟缓爱德华氏菌和副溶血弧菌这5种病原菌,使用该方法可以在1支反应管内快速、同步进行检测,得到大小分别为144、190、266、315和371 bp的特异性产物,其检出灵敏度分别为1.745、1.847、16.000、28.126和369.900 pg细菌基因组DNA;该方法特异性强,与大肠杆菌(Escherichia coli)、溶藻弧菌(V.alginolyticus)、河豚毒素假交替单胞菌(Pseudoalteromonas tetraodonis)、枯草芽孢杆菌(Bacillus subtilis)等其他细菌以及半滑舌鳎基因组DNA不产生交叉反应。2012年,应用该Arm-PCR方法对分离自病鱼的24个优势菌株进行了检测,确定出5株迟缓爱德华氏菌、3株嗜水气单胞菌、2株哈维氏弧菌及2株副溶血弧菌,证实该方法具有良好的可靠性和实用性。该方法不仅适用于海水养殖动物中致病性鳗弧菌、哈维氏弧菌、嗜水气单胞菌、迟缓爱德华氏菌、副溶血弧菌的快速、同步检测和病原流行情况调查,也为进一�Vibrio anguillarum, V. harveyi, Aeromonas hydrophila, Edwardsiella tarda and V. parahaemolyticus are five major bacterial pathogens isolated from marine aquaculture animals in China. In this report, five set of nested specific primers were designed based on the pathogenic factor genes and a specific amplicon rescue multiplex PCR （Arm-PCR） for the simultaneous detection of these five major bacterial pathogens were estab- lished. The concentration of primer Mix, Mg2＋, dNTPs, Taq DNA polymerase and the annealing temperature in the first step of Arm-PCR were optimized in this study. With summarizing the results of the experiment, in 50 ~tL of reaction volume, the optimized parameters were 10xPCR Buffer（20 mmol/L Mg2＋） 5 gL, dNTPs（2.5 mmol/L each） 5 gL, Taq DNA polymerase（2.5 U/gL） 0.6 pL, 10xprimer Mix（2 pmol/L） 5 gL, each template 1 gL. The annealing temperature was 55~C. The result showed that the Arm-PCR reported here could produce specific amplicons in one tube simultaneously. The expected sizes were 144, 190, 266, 315 and 371 bp for V. anguillarum, V. harveyi, A. hydrophila, E. tarda and V. parahaemolyticus, respectively. The sensitivities of the Arm-PCR were 1.745, 1.847, 16.000, 28.126 and 369.900 pg to above bacterial genomic DNA. There were no cross reactions with genomic DNA from healthy fish and other common bacteria, such as Escherichia coli, V. alginolyticus, Pseudoalteromonas tetraodonis, Bacillus subtilis, accorrding to the Arm-PCR. In 2012, the estab- lished Arm-PCR method was applied to the detection of 24 bacterial isolates from diseased fish and five strains of E. tarda, three strains of A. hydrophila, two strains V. harveyi and two strains V. parahaemolyticus were identified. The results showed that the method was reliable and practicable. The Arm-PCR method re- ported here can be used not only for the simultaneous specific detection and epidemiological survey of above five pathogens in marine aquacultural animals, but aslo for the development of microarray detection method

关 键 词：鳗弧菌 哈维氏弧菌 嗜水气单胞菌 迟缓爱德华氏菌 副溶血弧菌 套式PCR 检测 

分 类 号：S963.213[农业科学—水产养殖]