Panangala Victor S, Shoemaker Craig A, Van Santen Vicky L, Dybvig Kevin, Klesius Phillip H
Aquatic Animal Health Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, P.O. Box 952, Auburn, Alabama 36831-0952, USA.
Dis Aquat Organ. 2007 Mar 13;74(3):199-208. doi: 10.3354/dao074199.
A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 x 10(2) and 2.5 x 10(5) cells g(-1) of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.
开发了一种多重聚合酶链反应（m-PCR）方法，用于同时检测温水养殖中的3种重要鱼类病原体。通过调整反应缓冲液和采用降落式PCR程序，对用于从柱状黄杆菌（504 bp）、斑点叉尾鮰爱德华氏菌（407 bp）和嗜水气单胞菌（209 bp）扩增目标DNA片段的m-PCR进行了优化。每种细菌在每个m-PCR反应混合物中的最低检测限为20 pg核酸模板。在组织中检测这3种细菌的灵敏度阈值在3.4×10²至2.5×10⁵个细胞/克组织（斑点叉尾鮰Ictalurus punctatus Rafinesque）之间。用这3种细菌的10个代表性分离株以及在水生环境中分类学相关或普遍存在的11种其他革兰氏阴性菌和2种革兰氏阳性菌评估了m-PCR的诊断敏感性和特异性。除了一个单一物种（杀鲑气单胞菌杀鲑亚种）外，每组引物都能特异性扩增同源细菌物种的目标DNA。将m-PCR与细菌培养法进行比较，以鉴定实验感染鱼中的细菌。与耗时的传统细菌培养技术相比，m-PCR在快速、灵敏和同时检测感染鱼中的柱状黄杆菌、斑点叉尾鮰爱德华氏菌和嗜水气单胞菌方面似乎很有前景。
