| 摘要: |
| 【目的】建立可同时快速检测斑点叉尾鮰败血症3种病原（嗜水气单胞菌、维氏气单胞菌和鮰爱德华菌）的多重PCR检测方法。【方法】根据嗜水气单胞菌溶血素基因(Hly)、维氏气单胞菌脱氧核糖核酸酶Ⅰ基因(DNaseⅠ)以及鮰爱德华菌溶血活化基因(Eha)的保守序列，设计3对特异性引物，优化并建立斑点叉尾鮰败血症3种主要病原菌的多重PCR方法，对其特异性和灵敏度进行考察，并将其用于临床样品的检测。【结果】建立的多重PCR方法，当Hly基因、DNaseⅠ基因以及Eha基因的引物浓度分别为1.0，1.0和0.5 μmol/L，退火温度为59.5 ℃时，各目的片段均可较好地扩增。特异性试验结果表明，建立的多重PCR方法可从嗜水气单胞菌、 维氏气单胞菌和鮰爱德华菌分别扩增出1 091，262和450 bp的目的片段，从多种细菌DNA的混合种也可扩增出上述目的片段，而对其他对照组的扩增结果均为阴性；敏感性试验结果表明，建立的多重PCR最低可分别检测出200 CFU/mL的嗜水气单胞菌、300 CFU/mL的维氏气单胞菌和200 CFU/mL的鮰爱德华菌；对临床样本的检出率为100%，与传统生物学检测结果的符合率为100%。【结论】建立的多重PCR检测方法特异、灵敏、快速，可单独或同时检测斑点叉尾鮰败血症嗜水气单胞菌、维氏气单胞菌和鮰爱德华菌3种主要病原菌，也可以用于其他水生动物上嗜水气单胞菌、维氏气单胞菌和鮰爱德华菌的检测。 |
| 关键词: 斑点叉尾鮰 败血症 嗜水气单胞菌 维氏气单胞菌 鮰爱德华菌 多重PCR |
| DOI： |
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| 基金项目:国家科技支撑计划项目（2012BAD25B02）；现代农业产业技术体系建设专项(CARS-46) |
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| Establishment of multiplex PCR to detect three pathogens of “septicaemia” from channel catfish,Ictalurus punctatus |
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LIU Lihui,ZHANG Defeng,LI Ningqiu,et al
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| Abstract: |
| 【Objective】This study established a multiplex PCR method to rapidly detect three pathogens (Aeromonas hydrophila,Aeromonas veronii and Edwardsiella ictaluri) of “septicaemia” from channel catfish,Ictalurus punctatus.【Method】Three pairs of primers were designed based on the conservative sequences of hemolytic toxin genes (Hly) of A.hydrophila,deoxyribonucleaseⅠgenes (DNaseⅠ) of A.veronii and haemolysin activator genes (Eha) of E.ictaluri.Then multiplex PCR condition was optimized and the multiplex PCR for simultaneously detecting A.hydrophila,A.veronii and E.ictaluri was established.The specificity and sensitivity were detected and it was applied to clinical samples detection.【Result】All target fragments were amplified when the concentrations of Hly gene,DNaseⅠgene and Eha gene were 1.0,1.0 and 0.5 μmol/L and the annealing temperature was 59.5 ℃.The specificity test showed that fragments of 1 091, 262 and 450 bp were amplified from the genomic DNA of A.hydrophila,A.veronii and E.ictaluri,respectively.Three fragments were amplified from mixed DNA sample of A.hydrophila,A.veronii,E.ictaluri and other bacteria simultaneously,while no amplification was achieved from other negative control groups.The sensitivity test showed that the multiplex PCR had a high sensitivity with the detection limit of 200 CFU/mL for A.hydrophila,300 CFU/mL for A.veronii and 200 CFU/mL for E.ictaluri.The coincidence rate of the multiplex PCR method and traditional biology detection method for suspicious clinical samples was 100%.【Conclusion】The established multiplex PCR method was specific,sensitive and rapid to respectively or simultaneously detect the three pathogens of A.hydrophila,A.veronii and E.ictaluri of “septicaemia” from channel catfish,I.punctatus or other aquatic animals. |
| Key words: channel catfish septicaemia Aeromonas hydrophila Aeromonas veronii Edwardsiella ictaluri multiplex PCR |
